JP2008249422A - Concentration method of sample liquid, detection method of detection target substance and biochip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently concentrate even a trace amount of a sample liquid, and to reduce the errors caused by handling. <P>SOLUTION: The concentration method of the sample liquid containing a detection target substance includes a process for bringing a sample into contact with magnetic beads 14 to adsorb the medium other than the detection target substance in the sample liquid and a process for removing the magnetic beads 14 from the concentrated sample liquid, by applying a magnetic field to the magnetic beads 14. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sample liquid concentration method, a detection target substance detection method, and a biochip, and in particular, a detection target substance (biologically related substance) such as protein contained in a biological sample liquid such as blood is detected by an antigen-antibody reaction. It is related with the technique which acquires biological body related information by doing.

  In recent years, biosensing of antigen-antibody reaction using a minute space has been remarkably developed among analysis methods of trace substances related to clinical tests and environmental problems. In order to detect such a trace component with high accuracy, it is necessary to improve detection sensitivity.

  As a method for improving detection sensitivity, a method of concentrating a sample solution and increasing the concentration of a substance to be detected is a common method. For example, natural drying is simple and effective as a method for increasing the concentration of the detection target substance by evaporating the medium in the sample liquid. However, the evaporation rate of the medium is extremely slow, and it has been a problem that it takes too much time to concentrate a sample liquid containing a large amount of the medium.

On the other hand, for example, in Patent Document 1, a nonionic water-soluble monomer containing 60 mol% or more of N-vinylcarboxylic amide and 0.1 to 10 mol% of polyvalent vinyl with respect to the nonionic water-soluble monomer. It has been proposed to use a water-absorbing resin for concentrating biological fluids, which is a dried product of a polymer obtained by copolymerizing compounds. It is described that by introducing this water-absorbing resin for concentrating biological fluid into a test solution, the test solution can be concentrated, and adsorption / fixing action derived from hydrogen bonding (binding with protein, etc.) can be eliminated. The concentrated biological fluid is separated and recovered from the water absorbent resin by centrifugal filtration or suction filtration and analyzed.
JP 2000-72222 A

  However, when the concentration is performed at a place different from the analyzer as in the method of Patent Document 1, the concentrated sample solution is collected by filtration and then introduced into the analyzer using a pipette or a syringe. There was a need. At this time, if there is an error due to handling or the like in the amount of the sample solution to be introduced, the influence on the amount of the analysis component is large, and an error is likely to occur in the analysis data. In particular, when only a very small amount of sample liquid can be collected, such as a biological fluid, there is a problem that the influence of the error becomes large. Moreover, the trouble of introducing the concentrated sample solution into the analyzer is complicated, and the entire analysis time including pretreatment may increase.

  In addition, when the detection target substance contained in the sample liquid is generated by the reaction, for example, when the antigen-antibody reaction is caused by contacting the biological fluid containing the antigen with the antibody and the product obtained thereby is detected, the reaction time is short. In order to generate the antigen-antibody reaction described above, it is necessary to increase the antigen concentration in the sample solution.

  The present invention has been made in view of such circumstances, and a sample liquid concentration method and a detection target substance detection method capable of efficiently concentrating even when the sample liquid is very small and reducing errors due to handling and the like. And it aims at providing a biochip.

  Claim 1 of the present invention is a method for concentrating a sample liquid containing a detection target substance in order to achieve the above object, wherein the sample liquid and magnetic beads are brought into contact with each other to detect the substance other than the detection target substance in the sample liquid. There is provided a sample liquid concentration method comprising: adsorbing a medium; and removing the magnetic beads from the concentrated sample liquid by applying a magnetic field to the magnetic beads.

  According to the first aspect, since the step of bringing the magnetic beads into contact with the sample liquid and adsorbing a medium other than the detection target substance in the sample liquid is performed, the concentration of the detection target substance contained in the sample liquid can be increased. In addition, the concentrated sample solution is removed from the sample solution by moving the magnetic beads relatively by the action of a magnetic field, so the concentrated sample solution is filtered or collected with a syringe or the like. Even if it does not do, it can isolate | separate only the concentrated sample liquid. Therefore, even when the sample solution is very small, it is possible to efficiently concentrate the detection target substance and reduce errors such as handling.

  Here, the method of applying the magnetic field is not particularly limited, and examples thereof include a method of bringing a magnet close to the sample solution, a method of dropping a sample solution on a substrate on which a coil is arranged, and causing a current to flow through the coil. It is done.

  Magnetic beads are particles with a coating layer that adsorbs medium components other than the substance to be detected around the core particles with magnetism made of iron, nickel, cobalt, or their oxides. Say. Examples of the material for the coat layer include water-soluble polymers. Although it does not specifically limit as a sample liquid, For example, biological fluids, such as blood, saliva, nasopharyngeal fluid, breast milk, are contained. The medium is not particularly limited as long as it is a sample liquid other than the detection target substance, and includes, for example, a solvent such as water.

  A second aspect of the present invention is characterized in that, in the first aspect, the amount of the sample solution is 20 μL or less.

  Thus, even when the sample solution is very small, the sample solution can be concentrated to the extent that the detection target substance can be detected with high accuracy.

  A third aspect of the present invention is characterized in that, in the first or second aspect, the magnetic beads are particles each including a magnetic particle and a coating layer that adsorbs the medium around the magnetic particle.

  According to the third aspect, since the magnetic beads include magnetic particles and a coat layer that adsorbs a medium other than the detection target substance around the magnetic particles, the medium other than the detection target substance in the sample liquid is provided. In addition to selective adsorption and removal, the magnetic beads can be moved relative to the sample liquid by the action of a magnetic field. Thereby, the droplets of the sample liquid can be concentrated and analyzed in a state where they are arranged in the detection area. Therefore, it is possible to suppress an error caused by handling or the like in the amount of the sample liquid.

  A fourth aspect of the present invention is characterized in that, in any one of the first to third aspects, the amount of concentration of the sample liquid is adjusted by changing the amount of magnetic beads brought into contact with the sample liquid.

  According to the fourth aspect, the amount to be concentrated can be adjusted with higher accuracy than the case where the concentration is performed only by natural drying. For this reason, the detection result can be obtained with good reproducibility.

  Claim 5 of the present invention includes the step of concentrating the sample solution by the sample concentration method according to any one of claims 1 to 4 and the sample solution after the concentration in order to achieve the object. And a step of detecting the detection target substance. A method for detecting the detection target substance is provided.

  According to the fifth aspect, the magnetic beads are moved and removed relative to the sample liquid, so that the concentrated sample liquid can be easily separated and recovered without taking out, and the substance to be detected in the concentrated sample liquid is further removed. Can be detected. Therefore, it is possible to suppress an error caused by handling or the like in the amount of the sample liquid.

  A sixth aspect of the present invention is the method according to the fifth aspect, wherein the concentrated sample solution contains an antigen, and in the detection step, an antibody is further contacted with the concentrated sample solution to cause an antigen-antibody reaction, and the antigen-antibody reaction The reaction product obtained by is detected.

  As described above, the present invention is particularly effective when detecting biologically relevant information in a biological fluid that can be collected only in a very small amount with high sensitivity.

  According to a seventh aspect of the present invention, in order to achieve the above object, the substrate surface is provided with a region where the magnetic beads and the sample liquid are brought into contact with each other, and a retreating portion for retracting the magnetic beads from the region. A biochip is provided.

  According to the present invention, even when the sample solution is very small, it can be efficiently concentrated, and errors due to handling can be reduced.

  Hereinafter, preferred embodiments of a sample liquid concentration method, a detection target substance detection method, and a biochip according to the present invention will be described in detail with reference to the accompanying drawings.

  First, a method for concentrating a sample solution according to the present invention will be described with reference to FIG. FIG. 1 is an explanatory diagram for explaining a time-series flow of the sample liquid concentration method according to the present invention, and FIG. 2 is a conceptual diagram for explaining the concept of magnetic beads. In this embodiment, an example in which a biological fluid containing an antigen is concentrated using magnetic beads and then brought into contact with an antibody preliminarily fixed on a substrate to cause an antigen-antibody reaction is described. However, the present invention is not limited thereto. Absent.

  First, as shown in FIG. 1A, an amount of dried magnetic beads 14 corresponding to the degree of concentration is placed on an antibody 12 that has been fixed to the substrate 10 by a predetermined amount in advance. A sample liquid L containing the antigen 16 is dropped. Examples of the biological fluid used in the present invention include blood, saliva, nasopharyngeal fluid, and breast milk. Details of the magnetic beads 14 will be described later.

  Next, as shown in FIG. 1B, the medium in the dropped sample liquid L is adsorbed to the magnetic beads 14 and, for example, a medium (water or the like) equivalent to its own weight is absorbed. Specifically, when the droplet of the sample liquid is 10 μL, 5 μL of the medium in the sample liquid L can be absorbed by introducing 5 mg of the magnetic beads 14. Thus, the magnetic beads 14 can adsorb only a medium other than the antigen (protein or the like) to be detected.

  Next, as shown in FIG. 1C, after the sample liquid L is concentrated to a predetermined ratio, a magnetic field is applied to the sample liquid L by the magnetic field applying means 18, and the magnetic beads 14 are removed from the sample liquid L. The removed magnetic beads 14 are retracted to the outside of the detection area (position where the sample liquid L is arranged).

  Then, as shown in FIG. 1D, the concentrated sample liquid L from which the magnetic beads 14 have been removed is brought into direct contact with the antibody 12 that has been fixed in advance on the substrate 10. Thereby, an antigen-antibody reaction is caused by the antigen 16 in the sample liquid L and the antibody 12 on the surface of the substrate 10, and the generated aggregates, reaction products, and the like can be optically analyzed. The removed magnetic beads 14 can be used repeatedly by drying.

  Thus, the sample solution can be concentrated by bringing the sample solution into contact with the magnetic beads 14. Further, after concentration, the magnetic beads 14 can be easily separated and recovered by removing them from the sample liquid without collecting the sample liquid with a pipette or the like. Therefore, even when the sample solution is very small, errors due to handling and the like can be reduced, and the antigen-antibody reaction can be performed with high efficiency.

  Further, unlike the method of evaporating the medium, the concentration method described above can adjust the amount to be concentrated according to the amount of magnetic beads 14 brought into contact with the sample liquid, so that concentration can be performed quantitatively and reproducibly.

  In addition, the inside of the sample liquid L can also be stirred by moving the magnetic beads 14 in small droplets within the sample liquid droplets by changing the magnetic field.

  As shown in FIG. 2, the magnetic beads 14 used in the present embodiment include those in which an adsorption layer 19b that adsorbs a medium is formed on the surface of the magnetic particles 19a.

  The magnetic particles 19a are not particularly limited as long as they have paramagnetism and receive the action of a magnetic field, and examples thereof include iron, nickel, cobalt, and oxides thereof.

  The adsorption layer 19b is not particularly limited as long as it can adsorb a medium other than the detection target substance, and examples thereof include polyethylene glycol (PEG) and acrylamide compounds. The adsorption layer 19b is not limited to one type, and may be two or more types.

  The particle size of the magnetic beads 14 is not particularly limited as long as the magnetic beads 14 can be operated by applying a magnetic field even in the sample solution, and for example, a particle having a particle size of about 4.5 μm can be used.

  The method for producing the magnetic beads 14 is not particularly limited. For example, the magnetic beads 19a are immersed on the surface of the magnetic particles 19a by various methods such as immersing the magnetic particles 19a in a polymer solution having the adsorptivity of the medium and drying them. The method etc. which provide a functional group etc. are mentioned.

Specific examples of the magnetic beads 14 include those obtained by coating finely crystallized iron oxide particles (Fe ₂ O ₃ , Fe ₃ O ₄ ) with a hydrophilic or hydrophobic polymer. This magnetic bead 14 can be produced by reacting a polyethylene glycol (PEG) with an amino group at its end to a bead (manufactured by Dynal) with an epoxy group exposed on the surface, and covalently bonding the epoxy group and the amino group. .

  Since the magnetic beads 14 have superparamagnetism but do not show permanent magnetism, the magnetism is redispersed without remaining. Further, since the magnetic particles 19a inside the magnetic beads 14 are protected from the outside by the adsorption layer 19b, they do not react excessively with the reagents in the droplets. Thereby, the magnetic beads 14 can selectively remove only the medium of the sample liquid.

  Note that the magnetic beads 14 are not limited to the embodiment shown in FIG.

  Further, the magnetic field applying unit 18 is not particularly limited as long as it can apply a magnetic field, and examples thereof include a magnet and an electromagnetic coil.

  Next, an example to which the sample liquid concentration method according to the present invention is applied will be described.

  FIG. 3 is an explanatory diagram showing an example of a biochip to which the present invention is applied. Among these, FIG. 3 (A) is a perspective view, and FIG. 3 (B) is a cross-sectional view along the line AA. The present embodiment is a method of causing an antigen-antibody reaction by dropping a biological fluid (sample solution) containing an antigen into a biochip and bringing it into contact with an antibody preliminarily fixed in the biochip.

  As shown in FIG. 3, the biochip 20 includes a transparent flat substrate 24 in which a recess 22 for holding a sample liquid droplet is formed, a transparent cover plate 26 for covering the substrate 24, It has.

  On the substrate 24, recessed evacuation portions 28, 28 that communicate with the recess 22 and evacuate the magnetic beads 14 are formed. The retracting portion 28 may be any shape and size that can accommodate the magnetic beads 14 removed from the sample liquid in the recess 22, and is not limited to the form of FIG. 3.

  A supply port 30 for supplying the sample liquid to the recess 22 is formed on the surface of the cover plate 26 facing the recess 22, and can be closed by, for example, a seal member 32.

  As shown in FIG. 3B, the antibody 12 that specifically reacts with the antigen contained in the sample solution is fixed to the bottom surface of the recess 22 formed on the substrate 24. The method for fixing the antibody 12 is not particularly limited. For example, the antibody 12 can be formed by applying a sample containing the antibody. On the antibody 12, magnetic beads 14 for concentrating the sample solution are arranged. Thus, the recessed part 22 is an area | region which not only hold | maintains a sample liquid but can concentrate and detect.

  The substrate 24 and the cover plate 26 are integrated by being assembled by screws or the like (not shown) or by being in close contact with an adhesive.

  The sizes of the substrate 24 and the cover plate 26 are not particularly limited, but may be a portable size, for example, about 80 × 50 mm. The thicknesses of the substrate 24 and the cover plate 26 are not particularly limited, but can be about 5 mm, for example, in view of strength, economy, and the like.

  The material of the substrate 24 and the cover plate 26 is not particularly limited, but for example, a resin material is preferable. Moreover, it is preferable that the sample liquid in the biochip 20 is transparent because it enables visual and optical recognition of the sample liquid. As such materials, various resin plates, more specifically, various resins such as polydimethyl sulfoxide (PDMS), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), ultraviolet curable resin, polycarbonate (PC), etc. Various types of glass (soda lime glass, borosilicate glass, etc.) such as polyethylene terephthalate (PET), polyethylene naphthalate (PEN), and triacetyl cellulose (TAC) can be used.

  The biochip 20 configured as described above is set in, for example, an optical analyzer (not shown) including the magnetic field application unit 18, and the magnetic field application unit 18 is relatively relative to the surface of the substrate 24 of the biochip 20. By sliding, the magnetic beads 14 in the biochip 20 are removed from the sample liquid. Thereafter, the concentrated sample liquid in the recess 22 is analyzed by an optical analyzer (not shown).

  Thus, according to the present embodiment, the sample liquid can be concentrated by bringing it into contact with the magnetic beads 14. Further, after concentration, the sample solution can be easily separated by removing the magnetic beads 14 from the sample solution by applying a magnetic field without collecting the sample solution with a pipette or the like. Therefore, even when the sample solution is very small, errors such as handling can be reduced, and the antigen-antibody reaction can be performed with high efficiency.

  Moreover, the concentration method of this embodiment can adjust the amount to concentrate by the amount of the magnetic beads 14 brought into contact with the sample liquid, unlike the method of evaporating the medium. Therefore, analysis can be performed quantitatively and with good reproducibility.

  Next, a second embodiment according to the present invention will be described. This embodiment differs from the first embodiment mainly in that a plurality of sample liquid droplets are arranged on a substrate, and concentration and detection are performed on each droplet.

  FIG. 4 is a perspective view for explaining a second embodiment to which the present invention is applied. 4A is a perspective view illustrating a part of the biochip 40, and FIG. 4B is a cross-sectional view taken along the line BB.

  As shown in FIG. 4, the biochip 40 includes a substrate 42 on which a plurality of sample liquid droplets are arranged.

  On the substrate 42, the antibody 12 is fixed at a plurality of positions with predetermined intervals. Further, magnetic beads 14 are arranged on each antibody 12. In addition, in order to fix the magnetic beads 14 stably, a portion to which the antibody 12 is fixed may be formed in a concave shape with respect to the substrate 42 or may be given adhesiveness.

  The configuration of the magnetic beads 14, the method for manufacturing the magnetic beads 14, the method for fixing the antibody 12 to the substrate 42, and the like are almost the same as those in the first embodiment, and detailed description thereof is omitted.

  FIG. 5 is a diagram for explaining a time-series operation in the present embodiment.

  As shown in FIG. 5A, a sample liquid L of a biological fluid containing an antigen is dropped onto a substrate 42 on which a plurality of antibodies 12 of the same type are arranged with a predetermined interval. Then, the medium is adsorbed and concentrated by the magnetic beads 14 for each sample liquid L.

  Next, as shown in FIG. 5B, the magnetic field applying means 18 is disposed at a position corresponding to each sample liquid L on the back surface (the surface on which the sample liquid is not disposed) of the substrate 42. A magnetic field is applied to each sample liquid L by moving in the direction. Thereby, the magnetic beads 14 are removed from each sample liquid L.

  Then, as shown in FIG. 5C, an antigen-antibody reaction occurs between the antigen 16 contained in the sample liquid L and the antibody 12 fixed in advance on the substrate 42.

  As described above, according to the present embodiment, in addition to the same effects as those of the first embodiment described above, a plurality of sample liquids can be concentrated and reacted at the same time. Can be carried out at once.

  The preferred embodiments of the sample liquid concentration method, the detection target substance detection method, and the biochip according to the present invention have been described above, but the present invention is not limited to the above-described embodiments, and various aspects can be adopted. .

  For example, in each of the above-described embodiments, a liquid sample containing a biological substance such as an antigen or an antibody is used as the sample liquid.

  In each of the above embodiments, the method has been described in which the antibody is fixed on the substrate in advance, and the antigen-antibody reaction is performed by dropping the sample solution containing the antigen from the substrate. However, the present invention is not limited to this. After dropping on the sample, a sample containing the antibody can be added from above.

  In each of the above-described embodiments, the magnetic beads 14 that mainly adsorb a medium such as moisture have been described. However, the present invention is not limited to this, and magnetic beads having a portion that selectively adsorbs a protein or the like unnecessary for analysis can also be used. .

  In each of the above embodiments, as a method of driving the magnetic beads 14, a method of applying a magnetic field by arranging a coil on the substrate body and passing a current through the coil can also be adopted.

  In each of the above embodiments, the example in which the magnetic beads 14 are used for the purpose of concentrating the sample liquid by adsorbing the medium has been described. However, the present invention is not limited to this. For example, a substance involved in the reaction is added to the surface of the magnetic beads 14. It can also be used for the purpose of promoting the reaction by forming and increasing the contact efficiency with the components in the sample liquid.

It is explanatory drawing explaining the time-sequential flow of the concentration method of the sample liquid which concerns on this invention. It is a conceptual diagram explaining the concept of the magnetic bead in FIG. It is explanatory drawing which shows 1st Embodiment of the biochip to which this invention is applied. It is a perspective view which shows 2nd Embodiment of the biochip to which this invention is applied. It is explanatory drawing which shows the effect | action of 2nd Embodiment.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Board | substrate, 12 ... Antibody, 14 ... Magnetic bead, 16 ... Antigen, 18 ... Magnetic field application means, 19a ... Magnetic particle, 19b ... Adsorption layer, 20, 40 ... Biochip, 22 ... Recessed part, 24, 42 ... Substrate, 26 ... Cover plate, 28 ... Retraction part

Claims (7)

A method for concentrating a sample liquid containing a detection target substance,
Adsorbing a medium other than the detection target substance in the sample liquid by bringing the sample liquid and magnetic beads into contact with each other;
Removing the magnetic beads from the sample solution by applying a magnetic field to the magnetic beads;
A method for concentrating a sample solution, comprising:   The method for concentrating a sample liquid according to claim 1, wherein the amount of the sample liquid is 20 μL or less.   3. The method for concentrating a sample liquid according to claim 1, wherein the magnetic beads are particles each having a magnetic particle and a coating layer that adsorbs the medium around the magnetic particle.   The method for concentrating a sample solution according to any one of claims 1 to 3, wherein the amount of concentration of the sample solution is adjusted by changing the amount of magnetic beads brought into contact with the sample solution. A step of concentrating the sample liquid by the sample concentration method according to claim 1;
Detecting the detection target substance contained in the sample liquid after the concentration;
A method for detecting a substance to be detected, comprising: The concentrated sample solution contains an antigen,
6. The detection target according to claim 5, wherein in the detection step, an antibody is further brought into contact with the concentrated sample solution to cause an antigen-antibody reaction, and a reaction product obtained by the antigen-antibody reaction is detected. Substance detection method. On the substrate surface,
A region where the magnetic beads and the sample liquid are brought into contact with each other;
A retracting portion for retracting the magnetic beads from the region;
A biochip characterized by comprising:

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