JP2008275523A - Liquid sample analysis apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid sample analysis apparatus and its measuring method causing no particle concentration gradient in sample liquid because an analytical reagent is uniformly dissolved and dispersed in the sample liquid, in the apparatus for analyzing a specific substance in the sample liquid using particles carrying a reagent such as an antibody, as the analytical reagent. <P>SOLUTION: The liquid sample analysis apparatus includes (A) a liquid sample analysis chip including a space for filling the liquid sample, an analytical reagent holding part provided on the internal wall surface of the space, and an analysis part, (B) a magnetic field generating unit provided outside the liquid sample analysis chip, and (C) an analyzing unit provided outside the liquid sample analysis chip. The analytical reagent holding part has magnetic particles carrying a substance uniquely bonded to a specific component in the liquid sample, and the magnetic field generating unit is arranged so that the particles can be fixed to the analytical reagent holding part when filling the liquid sample in the analytical reagent holding part. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to analysis of a specific substance in a liquid sample, and particularly to an analyzer using an immunological reaction.

  In fields such as medicine, it is useful to analyze the presence and amount of a specific substance in a living body. Among them, an immunological analysis method using an antibody that specifically reacts with a substance to be analyzed is one of the most frequently used methods. There are various measurement methods for immunological analysis depending on the detection method of the reaction. For example, a labeled immunoassay method is known in which a reagent in which a labeling substance is linked to an antibody is used to detect whether or not an antigen-antibody reaction has occurred using the labeling substance. As this method, a method using an enzyme, a fluorescent substance, and a radioisotope as a labeling substance is known. These are called enzyme immunoassay, fluorescent substance-labeled immunoassay, and radioimmunoassay, respectively. Further, an immunoassay method based on an agglutination reaction is known in which a particulate reagent carrying an antibody is used to detect whether an antigen-antibody reaction has occurred or not based on the agglutination state of the particulate reagent. A typical example is a latex agglomeration method using latex particles.

  Although the labeled immunoassay can perform highly accurate analysis, there are problems that the reagent used is unstable and the measurement time is relatively long. On the other hand, the latex agglutination method, which is an immunoassay based on the agglutination reaction, has the advantage that the measurement time is relatively short, although the analysis accuracy is not so high.

  The latex agglomeration method is performed as follows. First, an antibody that specifically reacts with a substance to be analyzed is supported (fixed) on latex particles. The latex particles and the sample are reacted, and the substance to be analyzed contained in the sample is reacted with the antibody supported on the latex particles (antigen-antibody reaction). Latex particles aggregate as the reaction proceeds, so the aggregate is optically detected and the concentration of the analyte is measured.

  The measurement accuracy and measurement time in this method vary depending on the particle size of the latex particles used. If the latex particle size is large, the measurement accuracy is high, but the measurement time is long. If the particle size is small, the measurement accuracy is low, but the measurement time is short. Usually, the particle diameter of latex particles used in the method is several hundred nm, and the measurable region is about nM.

  Several improved methods have been reported for the latex agglomeration method. Patent Document 1 discloses a method in which magnetic particles having a particle size of 200 to 2 μm are used instead of latex particles, and an external magnetic field is applied to promote aggregation when the particles aggregate in a sample solution. According to this method, the measurement can be performed in about 10 minutes.

Patent Document 2 discloses a method of arranging particles in a straight line by using latex particles having a particle diameter of 0.5 μm to 10 μm and applying an electric field by applying an AC voltage to the sample solution. According to this method, 90% of all particles can be aggregated in one minute.
JP-A-62-287159 Japanese Patent Laid-Open No. 7-83928

  As described above, several methods have been reported so far for improving the aggregation rate of particles in an immunoassay performed by agglutinating antibody-carrying particles and the like. However, the immunoassay has a problem that the concentration of the substance to be measured cannot be accurately calculated if there is variation in the concentration of antibody-carrying particles in the liquid sample before the aggregation reaction. In the immunoassay method, since a plurality of antibodies are usually carried on a particle, one particle can react with a plurality of antigens. However, when antibody-carrying particles are dispersed non-uniformly in a liquid sample, the amount of antigen that can react with one particle is larger in a region where the particle concentration is low than in a region where the particle concentration is high. That is, an imbalance occurs in the amount of antigen bound to one particle. If the aggregation of particles is promoted in such a state, the analysis accuracy decreases because the aggregation rate varies locally.

  In order to improve the analysis accuracy, the inventors require that particles carrying a substance such as an antibody are uniformly dispersed and dissolved in the sample solution before the aggregation reaction. I found.

  By the way, in an analysis chip using particles carrying a substance such as an antibody as a reagent, the particles may be placed on the analysis chip in a dry state in order to maintain the storage stability of the reagent. The above-described problem of deterioration in analysis accuracy is particularly significant in such a case. In an analysis chip in which the particles are arranged in a dry state, the particles are dissolved and dispersed in the liquid when a liquid sample is introduced into the chip. At this time, since the particles flow into the inflowing liquid, there is a difference in the concentration of the particles between the front end and the end of the flow. When the particles are dissolved in the sample solution and the dissolution rate is significantly slower than the flow rate of the sample solution, it is less likely to be a problem. Such cases are rare.

  As described above, the present invention is an apparatus for analyzing a specific substance in a sample solution using an analysis reagent containing particles carrying an antibody or the like, and the particles are uniformly dissolved and dispersed in the sample solution. An object of the present invention is to provide a liquid sample analyzer that does not generate a particle concentration gradient and a measuring method thereof.

  As a result of intensive studies, the present inventors have completed the present invention by focusing on controlling the movement of the particles by applying a magnetic field from the outside using the particles as magnetic particles. That is, the said subject is solved by the following liquid sample analyzer and analysis method of this invention.

[1] (A) a space for filling a liquid sample; and an analysis reagent holding unit provided on an inner wall surface of the space; and a liquid sample analysis chip including the analysis unit; and (B) an outside of the liquid sample analysis chip And (C) a liquid sample analyzer including an analysis unit provided outside the liquid sample analysis chip,
The analysis reagent holding unit has magnetic particles carrying substances that specifically bind to specific components in a liquid sample,
A liquid sample analyzer arranged so that the magnetic field generating unit can fix the particles to the analysis reagent holding unit when the analysis reagent holding unit is filled with the liquid sample.
[2] The liquid sample analyzer according to [1], wherein the magnetic field generation unit is disposed so as to apply a magnetic field to all of the analysis reagent holding unit.
[3] The liquid sample analyzer according to [1] or [2], wherein the magnetic field generation unit can adjust the strength of a magnetic field applied to the particles.
[4] The liquid sample analyzer according to any one of [1] to [3], wherein the magnetic field generation unit includes a magnet.
[5] The liquid sample analyzer according to any one of [1] to [4], wherein the particles are placed in an analysis reagent holding unit in a dry state.
[6] The liquid sample analyzer according to any one of [1] to [5], wherein the analysis reagent holding unit and the analysis unit of the liquid sample analysis chip are provided at the same site in the space.
[7] The analysis reagent holding part and the analysis part of the liquid sample analysis chip are provided at different sites in the space, and the analysis reagent holding part and the analysis part are connected by a flow path [1] ] The liquid sample analyzer according to any one of [5] to [5].
[8] The liquid sample analyzer according to any one of [1] to [7], wherein the analysis unit includes an electrode pair.
[9] The liquid sample analyzer according to any one of [1] to [8], wherein the analysis unit includes an optical analyzer.
[10] The liquid sample analyzer according to any one of [1] to [9], wherein the analysis unit includes a magnetic measuring instrument.
[11] The liquid sample analyzer according to any one of [1] to [10], wherein the substance that specifically binds to a specific component in the liquid sample carried on the particles is an antibody.
[12] An analysis method using the liquid sample analysis chip according to any one of [1] to [11], wherein (a) a magnetic field generated from the magnetic field generation unit is applied to cause the particles to move in the space. A step of fixing to the inner wall surface, (b) a step of filling a liquid sample into the space in the liquid sample analysis chip from the liquid sample inlet, and (c) an action of the magnetic field generated from the magnetic field generating unit on the particles. An analysis method comprising: weakening and releasing the particles fixed to the inner wall surface of the space into the liquid sample; and (d) analyzing the liquid sample with the analysis unit.

  The present invention relates to an apparatus for analyzing a specific substance in a sample solution using an analysis reagent containing magnetic particles carrying a substance that specifically binds to a specific component in a liquid sample. Provided are a liquid sample analyzer that uniformly dissolves and disperses and does not generate a particle concentration gradient in a sample liquid, and a measurement method using the same. As a result, highly accurate analysis is possible.

1. Liquid sample analyzer of the present invention The liquid sample analyzer of the present invention is
(A) a space for filling a liquid sample; and an analysis reagent holding part provided on the inner wall surface of the space, and a liquid sample analysis chip including the analysis part,
(B) a magnetic field generation unit provided outside the liquid sample analysis chip, and (C) an analysis unit provided outside the liquid sample analysis chip,
When the analysis reagent holding part has magnetic particles carrying a substance that specifically binds to a specific component in the liquid sample, and the magnetic field generation unit is filled with the liquid sample in the analysis reagent holding part Further, the particles are arranged so as to be fixed to an inner wall surface of the space.

  The analysis target of the analyzer of the present invention is a liquid sample, more specifically, a specific substance present in the liquid sample. The liquid sample is not particularly limited, but examples thereof include blood collected from a living body.

  FIG. 1 is an exploded perspective view showing a first embodiment of the liquid sample analysis chip of the present invention. In the figure, reference numeral 1 denotes a liquid sample analysis chip, which includes an upper cover 21, a spacer 22, and a substrate 23. A liquid sample inlet 41 and an air hole 42 are formed in the upper cover 21. The spacer 22 has a hollow central portion in order to provide a space 3 for filling the liquid sample analysis chip 1 with the liquid sample. The space is connected to the outside by a liquid sample inlet 41 and an air hole 42.

FIG. 2 is a perspective view showing a liquid sample analyzer using the liquid sample analysis chip 1 shown in FIG. In the figure, 7 is a liquid sample analyzer, which includes a liquid sample analysis chip 1, a magnetic field generation unit 5, and an analysis unit 9. The magnetic field generation unit 5 includes a jackup system 51 that can adjust the distance between the unit and the liquid sample analysis chip 1 and a control device 52 that controls the jackup system 51. The analysis unit 9 includes an analysis device 91 and an analysis result output device 92. As shown in FIG. 2, the liquid sample analysis chip of this embodiment has a space 3 for filling the sample liquid. The space 3 includes an analysis reagent holding unit 31 and an analysis unit 32, and the entire space 3 becomes the analysis reagent holding unit 31 and the analysis unit 32.
The present invention will be described below with reference to the drawings. The description will be given with reference numerals in the figure as necessary.

1-1 (A) Liquid Sample Analysis Chip The liquid sample analysis chip is a minute member used for analyzing a liquid sample.
The liquid sample analysis chip of the present invention has a space for filling a liquid sample. An analysis reagent holding unit and an analysis unit are provided on the inner wall surface of the space. The liquid sample analysis chip is also simply called “chip”. The liquid sample analysis chip is preferably a rectangular parallelepiped.
(1) Space for filling liquid sample It is preferable that the space is provided in the center of the chip. The shape of the space is not limited, but is preferably a rectangular parallelepiped, for example. The liquid sample is preferably filled into the space by capillary action. It is known that the capillary phenomenon is affected by the shape of the space into which the liquid sample is introduced. For this reason, the space of the liquid sample chip preferably has a cross-sectional width of 0.5 to 5 mm and a height of 5 to 500 μm when the cross-section is square. The material forming the inner wall of the space is preferably a polymer film such as PET (polyethylene terephthalate) or polycarbonate. The surface of the material may be treated with a surfactant. Known surfactants are used.

(2) Analysis reagent holding part The liquid sample analysis chip of the present invention has an analysis reagent holding part provided on the inner wall surface of the space. The analysis reagent holding unit refers to a portion on the chip that holds an analysis reagent used for analyzing a liquid sample. An analysis reagent is disposed in the analysis reagent holding unit. In the analysis reagent holding unit, the liquid sample and the analysis reagent are mixed, and the analysis reagent is dissolved and dispersed in the liquid sample.

An analysis reagent is a reagent used for analyzing a liquid sample, and includes magnetic particles (also referred to as “supported particles” or simply “particles”) on which a substance that specifically binds to a specific component in the liquid sample is supported. The “substance that specifically binds to a specific component in a liquid sample” refers to a substance that can recognize a specific part of a specific component (analyte) in the liquid sample and bind to the part. Examples of such substances include biopolymers such as antibodies, antigens, receptors, enzymes, nucleic acids, peptides.
An antibody is a substance that can bind to a specific antigen in a liquid sample. Known antibodies can be used. Examples include anti-albumin antibody, anti-HCG antibody, anti-IgA antibody, anti-IgM antibody, anti-IgE antibody, anti-IgD antibody, anti-AFP antibody, anti-DNT antibody, anti-prostaglandin antibody, anti-human clotting factor antibody, anti-CRP Antibodies, anti-HBs antibodies, anti-human growth hormone antibodies, anti-steroid hormone antibodies are included.
Examples of antigens include albumin, HCG, IgA, IgM, IgE, IgD, AFP, DNT, prostaglandin, human clotting factor, CRP, HBs, human growth hormone, steroid hormone.

The magnetic particles are particles made of a substance that can interact with a magnet. Examples thereof include magnetic metal powder, Fe ₃ O ₄ , γ-Fe ₂ 0 ₃ , Co-γ-Fe ₂ 0 ₃ , nylon, polyacrylamide and other composite fine particles of ferrite and ferrite. The particle diameter of the magnetic particles is preferably several hundred nm to 10 μm. In the present invention, “to” includes numerical values at both ends thereof.

  “The magnetic particles carry a substance that specifically binds to a specific component in the liquid sample” means that the substance is chemically or physically bound to the surface of the magnetic particles. For example, the substance and magnetic particles can be obtained by chemically bonding using a silane coupling agent or the like. Moreover, you may use the commercially available magnetic bead which has an epoxy group, a tosyl group, an amino group, and a carboxylic acid group on the surface.

  The present analysis reagent may contain other reagents in order to enhance the storage stability of the “substance that specifically binds to a specific component in the liquid sample”. Examples of such reagents include saccharides, BSA, and commercially available protein stabilizers.

  In the present invention, the “substance that specifically binds to a specific component in a liquid sample” is preferably an “antibody”. Therefore, unless otherwise specified, the present invention will be described by taking a liquid sample analyzer using an analytical reagent containing “magnetic particles carrying antibodies” as an example.

  In the present invention, when the antigen A, which is the analyte in the liquid sample, has a plurality of recognition sites for antibodies, a plurality of supported particles can be bound to one A. Therefore, the supported particles are aggregated, and the analyte in the sample liquid is quantified from the degree of aggregation. In this case, it is sufficient that one or more antibodies are supported on the supported particles.

  On the other hand, when the antigen B as the analyte in the liquid sample has only one recognition site for the antibody, the antigen C has a plurality of recognition sites capable of binding to the antibody together with the magnetic particles carrying the antibody. Also, it is preferable that the analysis reagent is placed in the analysis reagent holding part. When the analyte B does not exist at all in the sample solution, the supported particles can aggregate with each other because they can bind to a single antigen C. However, if the analyte B is present in the liquid sample, the binding / dissociation between B and the supported particles and the binding / dissociation between C and the supported particles occur in equilibrium. At this time, since only one supported particle can be bonded to one B, the supported particles bonded to B cannot be aggregated. Therefore, the degree of agglomeration of the supported particles is smaller than when B is not present at all. That is, the analysis object B can be quantified by the decrease in the degree of aggregation.

The analysis reagent is disposed in the analysis reagent holding unit. A method for arranging the analysis reagent in the analysis reagent holding unit will be described later, but the arranged analysis reagent is preferably in a dry state. This is because the storage stability of the analysis reagent can be enhanced.
The liquid sample analysis chip of the embodiment shown in FIGS. 1 and 2 has an analysis reagent holding unit 31 and an analysis unit 32 at the same site in the space 3 of the chip. Further, the liquid sample analysis chip of the embodiment shown in FIG. 5 has the analysis reagent holding unit 31 and the analysis unit 32 in different parts in the space 3.

(3) Analysis Unit The liquid sample analysis chip of the present invention includes an analysis unit provided in a space for filling a liquid sample. An analysis unit is a part for detecting physical and chemical changes of a liquid sample. In the present invention, aggregation of the supported particles occurs in the analysis section. Then, the analysis unit is analyzed by an analysis unit provided outside the liquid sample analysis chip, whereby the degree of aggregation of the analysis reagent is measured. Although analysis / analysis by the analysis unit will be described later, in the case where the aggregation of particles is analyzed by an optical method, the analysis unit is preferably a transparent member.

  Aggregation of the supported particles proceeds only by leaving them as they are, but it is known that when an electric field is applied to the particles supporting the antibody or the like, the particles are arranged in a straight line (Pearl Chain phenomenon) (Patent Document 2). ). Therefore, also in the present invention, it is possible to promote the aggregation of the particles by applying an electric field when the supported particles are aggregated. In order to apply an electric field to the liquid sample, it is preferable to provide an electrode pair in the analysis unit. An electrode pair is a conductor or semiconductor provided in pairs to create an electric field or to pass current.

FIG. 3 is an exploded perspective view showing a liquid sample analysis chip in which an electrode pair is arranged in the analysis unit. As shown in FIG. 3, the two conductors divided on the substrate 24 are arranged on a straight line.
In the figure, reference numeral 1 denotes a liquid sample analysis chip, which includes a substrate 24 provided with an upper cover 21, a spacer 22, and an electrode pair 8. The substrate 24 is longer than the upper cover 21 and the spacer 22, and has a “protruding portion” that protrudes when superimposed. The rest of the configuration is the same as the shape of the liquid sample analysis chip in the first embodiment. The electrode pair 8 is made of two linearly provided conductors. Each of the two conductors is parallel to the long side of the substrate 24 and extends from one end in the length direction of the substrate 24 to the other end. Each of the conductors is exposed (also referred to as “exposed portion”) in a space for filling the liquid sample of the liquid sample analysis chip. The two exposed portions are preferably provided in the vicinity of the two long sides of the space 3. The width of the conductor is preferably 0.1 to 5 mm.
The conductor existing in the protruding portion on the substrate 24 is connected to the voltage applying device 82 by the electrode connection terminal 81. In this way, a voltage is applied to the electrode pair 8.

In the chip shown in FIG. 3, the analysis unit 32 and the analysis reagent holding unit 31 are provided at the same site in the space 3 for filling the chip liquid. Therefore, the electrode pair is provided in the space 3.
On the other hand, as in the chip shown in FIG. 5, when the analysis unit and the analysis reagent holding unit are provided in different parts of the chip space 3, the electrode pair is provided in the analysis unit 32. In FIG. 5, the substrate 24 is longer than the upper cover 21 and the spacer 22, and has a “protruding portion” that protrudes when superimposed. The electrode pair 8 is composed of two linearly provided conductors. The two conductors are parallel to the long side of the substrate 24. Each of the two conductors has a start point corresponding to the left end of the analysis unit 32 on the substrate 24 and an end point on the right end of the substrate 24. Part of the two conductors is exposed to the analysis part (also referred to as “exposed part”). The two exposed portions are preferably provided in the vicinity of the side parallel to the long side of the substrate of the analysis unit 32. The conductor existing in the protruding portion on the substrate 24 is connected to the voltage applying device 82 by the electrode connection terminal 81. In this way, a voltage is applied to the electrode pair 8.

  3 and 5 can apply the electric field to the analysis part and thereby aggregate the supported particles. In addition, the particles can be aggregated even when a magnetic field is applied to the analysis unit using the supported particle magnetic field generating unit 5, and this method will be described later.

1-2 (B) Magnetic field generation unit The magnetic field generation unit refers to a device capable of generating a magnetic field. A magnetic field is a force field existing around a magnet or current, and is also called a magnetic field. In the present invention, when the analysis reagent holding part is filled with a liquid sample, it is used to fix the supported particles to the analysis reagent holding part which is a part of the inner wall of the space. Thereby, it is possible to prevent the supported particles from flowing by the liquid sample introduced into the analysis reagent holding unit. As a result, it is possible to avoid uneven distribution of the support particles in the sample liquid.

  When a liquid sample is introduced into the analysis reagent holding part in a state where the magnetic particles are applied and the supported particles are fixed to the analysis reagent holding part, most of the particles come into contact with the liquid sample while being restrained by the analysis reagent holding part. That is, the liquid sample and the supported particles are in contact with each other in a state where the distribution of the supported particles is not biased at the front end portion and the end portion of the liquid sample flow. Usually, a plurality of antibodies are supported on the supported particles. Therefore, a plurality of antigens can bind to one particle, but when contacting with a liquid sample in a state where the distribution of supported particles is not biased as described above, the number of antigens that can bind to one particle is almost the same in all particles. It is considered to be. Therefore, when the particles for measurement are aggregated, the degree of aggregation of the entire analysis unit can be made constant.

  However, as described above, when the distribution of the carrier particles in the liquid sample becomes non-uniform, a large number of antigens are likely to bind to one carrier particle at a low carrier particle concentration. On the other hand, where the carrier particle concentration is high, the number of antigens bound to one carrier particle decreases. The measurement accuracy decreases between the carrier particles to which a large amount of antigen is bound and the carrier particles to which only a small amount of the antigen is bound because the ease of aggregation of the carrier particles is different. The analyzer of the present invention can avoid such a problem.

  The magnetic field generating unit preferably includes a magnet. A magnet refers to a substance that is a source for generating a bipolar magnetic field. A known magnet may be used as the magnet. Examples of the magnet include a ferrite magnet, an alnico magnet, a neodymium magnet, and a member obtained by combining these with a polymer material (polymer). As shown in FIGS. 2, 4 and 6, the magnetic field generating unit is preferably arranged so that the magnetic field acts on the entire analysis reagent holding unit. Specifically, it is preferable that the shape and size be such that the entire analysis reagent holding part can be covered from the outside of the chip. This is because most of the supported particles present in the analysis reagent holding part can be fixed to the analysis reagent holding part. As shown in the figure, when the chip is a flat plate, the magnetic field generating unit is also preferably a flat plate.

  Furthermore, it is preferable that the magnetic field generation unit is provided with a jack-up system 51 so that the distance from the liquid sample analysis chip can be adjusted. Thereby, the strength of the magnetic field acting on the supported particles can be controlled. Further, the jack-up system 51 may control the distance and the like by the control system 52.

  The magnetic field generation unit may include an electromagnet. An electromagnet is a magnet that generates a magnetic field by an electric current flowing in a coil using an electromagnetic induction phenomenon. Examples of electromagnets include a soft iron core wound with a coil. An electromagnet generates a magnetic field when a current is passed, and disappears when the current is stopped. Therefore, the strength of the magnetic field acting on the carrier particles can be controlled without adjusting the distance from the tip.

  As described above, the magnetic field generation unit may act on the analysis unit to promote the aggregation of the supported particles. However, in this case, if the magnetic field generating unit is arranged so that the magnetic field can be applied to the entire analysis unit, the aggregation of particles is inhibited. Therefore, it is preferable that the magnetic field generating unit has a shape and a size such that the magnetic field acts only at a position where it is desired to aggregate the supported particles in the analysis unit. For example, if the magnetic field generating unit has a small circular shape and is installed only in a portion where the supported particles are to be aggregated in the analysis unit, the aggregation of the supported particles can be promoted locally.

1-3 (C) Analysis Unit The analysis unit is an apparatus that analyzes and analyzes changes in the liquid sample observed in the analysis unit on the chip and quantifies a specific substance in the liquid sample. Examples of means for analyzing changes in the liquid sample include optical techniques. Analyzing a liquid sample optically means analyzing the change behavior of the liquid sample by the absorbance and transmittance of the sample liquid with respect to light of a specific wavelength. Or it means judging the solid substance contained in the fixed area | region of an analysis part by external appearance property. Examples of measuring instruments for optically analyzing a liquid sample include an absorptiometer and an image processing apparatus. It may also be observed with the naked eye.

  In order to enable optical analysis of a liquid sample, the entire chip is preferably made of a transparent material. Examples of such materials include thermoplastic resins. Of these, polyethylene terephthalate and polycarbonate are preferable. In particular, polycarbonate is preferable because it has very high transparency to visible light.

  The analysis unit may include magnetic measurement means. Examples of the magnetic measurement means include a gauss meter. When the analysis unit is provided with a magnetic measuring means, the correlation between the strength of the magnetism and the degree of aggregation can be grasped when the supported particles are aggregated by applying magnetism. The strength of magnetism and the application time of magnetism required for making the supported particles into a certain agglomerate can be obtained, and the aggregation of the supported particles can be quantitatively analyzed.

2. Method for Producing Liquid Sample Dividing Device of the Present Invention The liquid sample dispensing device of the present invention can be arbitrarily produced within a range that does not impair the effects of the invention. A preferable production method will be described below.
2-1 (A) Manufacturing Method of Liquid Sample Analysis Chip As shown in FIGS. 1 and 3, the liquid sample analysis chip is preferably manufactured by bonding the upper cover 21, the spacer 22, and the substrate 23. For the upper cover 21 and the substrate 23, for example, a plastic material such as polycarbonate or polyethylene terephthalate, or glass can be used.
As the spacer 22, a double-sided adhesive sheet or an adhesive resin that can adhere the upper cover 21 and the substrate 23 can be used. Further, the spacer 22 may be made of the same material as the upper cover 21 and the substrate 23 and coated with an adhesive on the upper and lower surfaces.
As shown in FIG. 5, even when the analysis reagent holding unit 31 and the analysis unit 32 have a separated structure, they can be similarly manufactured.

  Arbitrary methods can be used as the method of arranging the analysis reagent at the site used as the analysis reagent holding part of the liquid sample chip. For example, a liquid (dispersion liquid) in which an analysis reagent is dispersed in water or the like may be prepared and injected from a liquid sample inlet 41 of a liquid sample analysis chip as shown in FIG. Specifically, the dispersion liquid is spotted on the liquid sample inlet 41 and it is confirmed that the liquid is filled in the analysis reagent holding unit 31. Next, the magnetic field generating unit 5 is brought close to the chip 1 so that the supported particles are fixed to the analysis reagent holding unit 31. In this state, the chip 1 and the magnetic field generating unit 5 are put into a dryer, and drying is performed until no liquid can be visually confirmed. In this way, it is possible to obtain a liquid sample analysis chip in which the dried supported particles are uniformly arranged in the analysis reagent holding unit 31.

You may apply | coat the said dispersion liquid to the part which comprises the space of the board | substrate 23 of a liquid sample analysis chip | tip shown in FIG. At this time, it is preferable to apply a magnetic field to the portion of the substrate 23 to which the liquid is applied using the magnetic field generating unit 5 because the supported particles can be uniformly arranged in the analysis reagent holding portion as described above. Alternatively, by applying the dispersion and performing lyophilization, the particles during loading can be arranged uniformly. A method for applying the liquid to the portion constituting the space of the substrate 23 is not particularly limited.
In addition to this, the dispersion may be dropped onto a portion of the chip constituting the space of the substrate 23 using a pipette and dried.
As shown in FIG. 5, when the analysis reagent holding unit 31 and the analysis unit 32 are separated from each other, the liquid is similarly applied to the bottom surface of the analysis reagent holding unit 31 of the substrate 24 or dried after being dropped. It is preferable to provide them.

  The method of providing the electrode pair 8 on the substrate 24 as shown in FIGS. 3 and 5 is not particularly limited. For example, a paste containing a conductor may be printed on the substrate 24, or a conductor material or the like may be formed on the substrate 24 by sputtering or vapor deposition. Examples of the conductor material include gold, platinum, silver, copper, palladium, chromium, and carbon. It is preferable that the material of the conductor does not ionize due to electrochemical reaction in the sample solution or undergo oxidation or reduction.

2-2 (B) Magnetic field generation unit A magnetic field generation unit can be manufactured using a well-known thing. For example, a magnetic field generating unit can be produced by processing a ferrite magnet into a flat plate having a desired size. A polymer sheet in which ferrite particles are dispersed in a polymer may be processed into a desired size to form a magnetic field generating unit. Furthermore, an electromagnet may be produced by winding a conductive wire around soft iron of a desired size to form a magnetic field generating unit.
Known jackup systems 51 and control devices 52 used together with the magnetic field generating unit can also be used.

2-3 (C) Analysis Unit The analysis unit 9 can also be obtained using a known absorbance meter or camera. The analysis unit may be the examiner's naked eye. The analysis device 91 and its output device 92 can be a computer with built-in image processing software.

3. Analysis method using the liquid sample analyzer of the present invention Analysis method using the liquid sample analyzer of the present invention,
(A) a step of fixing a magnetic particle (supported particle) carrying a substance that specifically binds to a specific component in a liquid sample by applying a magnetic field generated from the magnetic field generating unit to the inner wall surface of the space;
(B) filling the liquid sample into the space in the liquid sample analysis chip from the liquid sample inlet;
(C) a step of weakening the action of the magnetic field generated from the magnetic field generating unit on the supported particles and releasing the supported particles fixed to the inner wall surface of the space into the liquid sample; and (d) by the analysis unit Preferably including a step of analyzing the liquid sample.

First, an analysis method using the liquid sample analysis chip shown in FIG. 2 will be described.
As described above, the step (a) can be performed by bringing the magnetic field generating unit closer to the chip. Alternatively, when the magnetic field generating unit is an electromagnet, a current may be passed to cause the electromagnet to generate a magnetic field. The strength of the magnetic field may be any strength as long as the supported particles in the analysis reagent can be fixed to the analysis reagent holding portion.

  Next, the liquid sample is filled in the chip by the step (b). The method for introducing the liquid sample into the chip is not particularly limited. It is preferable that a liquid sample is spotted on a liquid sample inlet provided in the chip and is filled using a capillary phenomenon. After filling, it is preferable that the liquid sample and the supported particles are allowed to stand for a certain period of time in order to sufficiently contact them.

  Subsequently, the action of the magnetic field on the supported particles is weakened by the step (c), and the supported particles fixed to the inner wall surface of the space are released into the liquid sample. To weaken the action of the magnetic field on the supported particles means to make the strength that the supported particles cannot be fixed to the analysis reagent holding unit, or to make the magnetic field zero. When the magnetic field generating unit is a magnet or the like, the magnetic field can be weakened by separating the distance from the chip. In this case, it is sufficient if the strength is such that the supported particles cannot be fixed to the analysis reagent holding part even if the magnetic field is not completely zero. When the magnetic field generating unit is an electromagnet, the magnetic field can be reduced to zero by setting the current to zero. The released carrier particles begin to aggregate with each other.

  Next, in the step (d), the liquid sample is analyzed using the aforementioned analysis unit. For example, the size or the like of the aggregate is observed by the analysis unit 9 and the aggregate formation rate is calculated by the analyzer 91, and the concentration of the analyte in the sample is calculated from this value. The analysis result is output from the output device 92. In this way, a liquid sample can be analyzed with high accuracy.

  Next, an analysis method using the liquid sample analysis chip shown in FIG. 4 will be described. When the liquid sample analysis chip in FIG. 4 is used, the steps (a) to (c) are performed in the same manner as in the liquid sample analysis chip shown in FIG. However, in step (d), an AC potential is applied to the electrode pair 8 through the electrode connection terminal 81 by the potential applying device 82. As a result, the supported particles 6 aggregate so as to form a pearl chain (see Patent Document 2), and thus the aggregation of the supported particles can be promoted.

  When the liquid sample analysis chip of FIG. 6 is used, the analysis reagent holding unit 31 and the analysis unit 32 are provided separately, and therefore the steps (a) to (c) are performed by the analysis reagent holding unit 31. Then, a liquid sample is transferred to the analysis part 32 and (d) process is performed. Specifically, it is preferable to perform analysis by the following method.

1) First Method An opening is provided in the flow channel 33, and the inner wall of the flow channel between the analysis reagent holding unit 31 and the analysis unit 32 is discontinuous. A magnetic field is applied while the opening is open to fix the supported particles to the inner wall surface of the chip (step a). Next, a liquid sample is spotted on the liquid sample inlet 41 provided on the chip, and the liquid sample is filled into the space 3 of the chip (step b). The amount of the liquid sample deposited on the liquid sample inlet 41 is equal to or greater than the total capacity (also referred to as “space capacity”) of the analysis reagent holding unit 31, the analysis unit 32, and the flow path 33. The liquid sample penetrates to the analysis reagent holding part 31 by capillary action, but cannot penetrate since the discontinuous part exists in the flow path thereafter. This phenomenon is generally called “capillary valve phenomenon”. At this time, the liquid sample rises from the liquid sample inlet 41 and overflows. In this state, the action of the magnetic field on the supported particles is weakened, and the liquid sample and the supported particles are sufficiently brought into contact (step c). Thereafter, the opening is closed, the discontinuous structure of the flow path is eliminated, and the liquid sample is transferred from the analysis reagent holding part 31 to the analysis part 32 via the flow path 33 by capillary action. Then, analysis is performed (step d).

  The opening may be provided with a lid that fits into the opening. At this time, it is preferable that a part of the lid forms a hinge (hinge) with the wall surface. The opening can be closed by the lid.

2) Second method A magnetic field is applied to fix the supported particles to the inner wall surface of the chip (step a). A liquid sample having a volume equal to the volume of the analysis reagent holding unit 31 is spotted on the liquid sample inlet 41 (step b). Then, the liquid sample is filled into the analysis reagent holding unit 31 and cannot enter further. In this state, the action of the magnetic field on the supported particles is weakened, and the liquid sample and the supported particles are sufficiently brought into contact (step c).
Thereafter, a liquid sample equal to or larger than the capacity of the analysis unit 32 is spotted on the liquid sample inlet 41, and the liquid sample is transferred from the analysis reagent holding unit 31 to the analysis unit 32 via the flow path 33. Then, analysis is performed (step d).

3) Third method A magnetic field is applied to fix the supported particles on the inner wall surface of the chip (step a). With the air hole 42 opened, a liquid sample larger in volume than the space capacity is spotted on the liquid sample inlet 41, and the liquid sample is introduced into the chip space 3 (step b). The air hole 42 is closed when the analysis reagent holding unit 31 is filled with the liquid so that the liquid sample does not enter the flow path 33. At this time, the liquid sample rises from the liquid sample inlet 41 and overflows. In this state, the action of the magnetic field on the supported particles is weakened, and the liquid sample and the supported particles are sufficiently brought into contact (step c). Thereafter, the air hole 42 is opened, and the liquid sample is transferred from the analysis reagent holding unit 31 to the analysis unit 32 via the flow path 33 by capillary action. Then, analysis is performed (step d).
In this method, as described in the first method, a lid that fits into the air hole 42 may be provided to open and close the air hole 42.

4) Fourth Method A magnetic field is applied to fix the supported particles on the inner wall surface of the chip (step a). A liquid sample having the same volume as the analysis reagent holding unit 31 is spotted at the liquid sample inlet 41, and the liquid sample is filled into the space 3 of the chip (step b). Then, since the liquid is filled in the analysis reagent holding part 31, the action of the magnetic field on the supported particles is weakened in this state, and the liquid sample and the supported particles are sufficiently brought into contact (step c). Thereafter, the liquid sample is transferred to the analysis unit 32 by centrifugal force. Then, analysis is performed (step d).

5) Fifth Method A magnetic field is applied to fix the supported particles on the inner wall surface of the chip (step a). A pump is connected to the liquid sample inlet 41, and the liquid sample is filled into the analysis reagent holding unit 31 by the pump (step b). In this state, the action of the magnetic field on the supported particles is weakened, and the liquid sample and the supported particles are sufficiently brought into contact (step c). Thereafter, the liquid sample is transferred to the analysis unit 32 by a pump. Then, analysis is performed (step d).

Hereinafter, the present invention will be described in more detail with reference to examples.
(Example 1: Analysis using the liquid sample analyzer shown in FIG. 2)
A substrate 23 made of polyethylene terephthalate having a thickness of 0.1 mm; a length of 20 mm; and a width of 10 mm was prepared. A double-sided adhesive tape having a thickness of 0.05 mm; a length of 20 mm; and a width of 10 mm was prepared and used as the spacer 22. An upper cover 21 made of polyethylene terephthalate having a thickness of 0.1 mm, a length of 20 mm, and a width of 10 mm with a hydrophilic treatment applied to the surface was prepared. A liquid sample inlet 41 and an air hole 42 were formed in the cover 21. These were bonded to obtain a liquid sample analysis chip 1 as shown in FIG. The chip has a space 3 for filling a liquid sample, and the analysis reagent holding unit 31 and the analysis unit 32 are the entire space 3.

  A commercially available anti-mouse IgG antibody-modified magnetic particle having a diameter of about 2.8 microns (Dynal, Dynabeads M-280) was loaded with a mouse-derived anti-human CRP (cyclic AMP receptor protein) monoclonal antibody. The operation was performed according to the protocol of the magnetic particle vendor. The magnetic particles were dispersed in 0.1 M glycine buffer containing 5% trehalose and 0.1% BSA. The liquid was spotted on the reagent inlet 2 of the liquid sample analysis chip 1 and it was confirmed that the liquid was filled in the space 3. Next, a magnet larger than the area of the space 3 was prepared, and the liquid sample analysis chip 1 was placed on the magnet. In this state, the liquid sample analysis chip 1 and the magnet were put into a dryer, and dried until the liquid component of the liquid filled in the space 3 could not be visually confirmed. Thereby, the liquid sample analysis chip 1 having the analysis reagent holding part 31 in which the anti-mouse IgG antibody-modified magnetic particles (supported particles) were uniformly dispersed in a dry state was prepared.

  With the liquid sample analysis chip 1 placed on the magnet, pure water was spotted on the liquid sample inlet 41 as a liquid sample, and it was confirmed that the liquid sample was filled in the space 3. Thereafter, the chip 1 was moved away from the magnet. As a result, the supported particles were dispersed in the liquid sample. The distribution of the supported particles in the liquid sample in the space 3 was observed with a microscope. As a result, it was confirmed that the supported particles were uniformly distributed in the liquid sample.

(Example 2)
The behavior of the supported particles was observed in the same manner as in Example 1 except that a solution containing CRP was used instead of pure water as the liquid sample. After the liquid sample was filled in the space 3, the chip 1 was moved away from the magnet to disperse the supported particles in the liquid sample, and the dispersion state was observed with a microscope. As a result, it was confirmed that the supported particles were aggregated in the liquid sample.

(Comparative Example 1)
The behavior of the supported particles was observed in the same manner as in Example 1 except that the liquid sample analysis chip 1 was not placed on the magnet. It was confirmed that when the pure water flows into the space 3, the supported particles 6 are caused to flow from the inner wall of the space 3 in which the supported particles 6 were disposed.

(Example 3: Analysis using the liquid sample analyzer shown in FIG. 4)
A polyethylene terephthalate substrate 24 having a thickness of 0.1 mm; a length of 20 mm; and a width of 10 mm was subjected to gold sputtering to provide two parallel conductors. The conducting wire was provided so that one end in the length direction of the substrate 24 was the starting point and the other end was the ending point. The conducting wire was provided in parallel with the long side of the substrate 24. The width of the conducting wire was 1 mm. The distance between the two conductors was 0.5 mm.
A double-sided adhesive tape having a thickness of 0.05 mm; a length of 20 mm; and a width of 10 mm was prepared and used as the spacer 22. An upper cover 21 made of polyethylene terephthalate having a thickness of 0.1 mm; a length of 20 mm; and a width of 10 mm was prepared. A liquid sample inlet 41 and an air hole 42 were formed in the cover 21. By bonding these together, a liquid sample analysis chip 1 as shown in FIG. 2 was obtained.

In the same manner as in Example 1, a liquid sample analysis chip 1 having an analysis reagent holding part 31 in which mouse IgG antibody-modified magnetic particles (supported particles) were uniformly dispersed in a dry state was prepared.
Using a solution containing CRP as the liquid sample, the liquid sample was filled in the space 3 in the chip while applying a magnetic field to the supported particles in the same manner as in Example 1. Subsequently, the application of the magnetic field was canceled in the same manner as in Example 1, and the supported particles were dispersed in the solution. Subsequently, the electrode connection terminal 81 was connected to the electrode pair 8 of the liquid sample analysis chip 1, and an AC voltage of 20 V was applied using the potential application device 82. As a result, it was confirmed that the aggregation of the supported particles was promoted.

  According to the present invention, an analysis reagent carrying an antibody or the like can be uniformly dissolved and dispersed in a sample solution, and a liquid sample can be analyzed without causing a concentration gradient in the sample solution. Therefore, the present invention is suitably used for an immunological analysis method using an antibody that specifically reacts with a substance to be analyzed.

The disassembled perspective view which shows the 1st aspect of the liquid sample analysis chip | tip of this invention The perspective view which shows the 1st aspect of the liquid sample analyzer of this invention The disassembled perspective view which shows the 2nd aspect of the liquid sample analysis chip | tip of this invention. The perspective view which shows the 2nd aspect of the liquid sample analyzer of this invention The disassembled perspective view which shows the 3rd aspect of the liquid sample analysis chip | tip of this invention. The perspective view which shows the 3rd aspect of the liquid sample analyzer of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Liquid sample analysis chip | tip 21 Cover 22 Spacer 23 Substrate 24 Substrate which has an electrode pair 3 Space 31 Analytical reagent holding | maintenance part 32 Analysis part 33 Flow path 41 Liquid sample inlet 42 Air hole 5 Magnetic field generation unit 51 Jackup system 52 Control apparatus 6 Supported particles 7 Liquid sample analyzer 8 Electrode pair 81 Electrode connection terminal 82 Potential application device 9 Analysis unit 91 Analysis device 92 Analysis result output device

Claims (12)

(A) a space for filling a liquid sample; and an analysis reagent holding part provided on the inner wall surface of the space, and a liquid sample analysis chip including the analysis part,
(B) A magnetic field generation unit provided outside the liquid sample analysis chip, and (C) a liquid sample analysis device including an analysis unit provided outside the liquid sample analysis chip,
The analysis reagent holding unit has magnetic particles carrying substances that specifically bind to specific components in a liquid sample,
A liquid sample analyzer arranged so that the magnetic field generating unit can fix the particles to the analysis reagent holding unit when the analysis reagent holding unit is filled with the liquid sample.   The liquid sample analyzer according to claim 1, wherein the magnetic field generation unit is disposed so as to cause a magnetic field to act on the entire analysis reagent holding unit.   The liquid sample analyzer according to claim 1, wherein the magnetic field generation unit can adjust the strength of a magnetic field applied to the particles.   The liquid sample analyzer according to claim 1, wherein the magnetic field generation unit includes a magnet.   The liquid sample analyzer according to claim 1, wherein the particles are arranged in the analysis reagent holding unit in a dry state.   The liquid sample analyzer according to claim 1, wherein the analysis reagent holding unit and the analysis unit of the liquid sample analysis chip are provided at the same site in the space.   The analysis reagent holding part and the analysis part of the liquid sample analysis chip are provided in different parts in the space, and the analysis reagent holding part and the analysis part are connected by a flow path. Liquid sample analyzer.   The liquid sample analyzer according to claim 1, wherein the analysis unit includes an electrode pair.   The liquid sample analyzer according to claim 1, wherein the analysis unit includes an optical analyzer.   The liquid sample analyzer according to claim 1, wherein the analysis unit includes a magnetic measuring instrument.   The liquid sample analyzer according to claim 1, wherein the substance that specifically binds to a specific component in the liquid sample carried on the particles is an antibody. An analysis method using the liquid sample analysis chip according to claim 1,
(A) applying a magnetic field generated from the magnetic field generating unit to fix the particles to the inner wall surface of the space;
(B) filling the liquid sample into the space in the liquid sample analysis chip from the liquid sample inlet;
(C) a step of weakening the action of the magnetic field generated from the magnetic field generating unit on the particles to release the particles fixed to the inner wall surface of the space into the liquid sample; and (d) a liquid by the analysis unit. Analyzing the sample.

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