JP2008232891A - Chip, apparatus, and method for measurement - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem that improvements in redispersibility after pearl chain formation has been required to directly measure specimens such as blood plasma since redispersibility after an A.C. electric field is shielded is greatly affected by viscosity due to diffusion and pearl chain formation is not released but as if in a held state when redispersibility is not satisfactory. <P>SOLUTION: A measuring chip is provided with: a liquid sample introduction means for introducing a liquid sample; a liquid sample holding means connected to the liquid sample introduction means to hold the liquid sample; and electrodes for applying an A.C. voltage to the liquid sample introduced to the liquid sample holding means. Part or a whole region of the liquid sample holding means of the measuring chip carries first particles and second particles. A first specific binding substance to a substance to be tested is immobilized to the first particles. The second particles have a capacity larger than that of the first particles, and a second specific binding substance to be specifically bound to the first specific binding substance immobilized to the first particles is immobilized to the second particles. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a measuring chip, a measuring apparatus, and a measuring method for measuring a test substance contained in a liquid sample. In particular, the present invention provides a system that can measure a measurement object contained in plasma obtained from blood without dilution.

  In recent years, it has become possible to measure various substances due to advances in analysis, analysis, and inspection technology. In particular, in the field of clinical testing, the development of measurement principles based on specific reactions such as biochemical reactions, enzyme reactions, and immune reactions has made it possible to measure substances in the blood that are reflected in the pathological condition.

  Among them, a clinical examination field called POCT (Point Of Care Testing) has attracted attention. POCT is intended to shorten the time from when a sample is collected until a test result is obtained, with simple quick measurement as the first. Therefore, what is required for POCT is a simple measuring principle, a measuring device that is small, portable, and easy to operate.

  With recent advances in technological development, for example, small measuring devices that can be easily measured, such as blood glucose sensors, have been developed. The ripple effect of POCT enables rapid and accurate diagnosis by acquiring rapid measurement results, as well as reducing the cost of testing, reducing the burden on subjects due to the small volume of blood samples, and infection It is possible to reduce the amount of radioactive waste. Furthermore, in today's medical settings, where “optimization of medical expenses”, “from treatment to prevention”, and “evidence-based medical care” are required to change, POCT devices are also expected to become important sources of information acquisition and transmission. ing. For the reasons described above, POCT-compatible measuring instruments have been developed to meet the needs shown here.

  The blood glucose sensor is a POCT-compatible measuring device that uses the enzyme reaction principle, but there are other POCT-compatible measuring devices that use the immune reaction principle. For example, there is a DCA2000 system which is a measurement device dedicated to hemoglobin A1c (HbA1c). The DCA2000 system measures HbA1c based on the latex immunoaggregation inhibition method, and includes HbA1c that has undergone pretreatment (hemolysis / denaturation treatment), anti-HbA1c monoclonal antibody immobilized on latex beads, and an epitope unique to HbA1c. The amount of latex agglutinated complex that is prevented from being produced is detected by the transmitted light intensity according to the HbA1c concentration. As a system, the reagent shown here is provided in a dedicated reaction cassette in a state where the reagent is supported and a diluent is enclosed. The latex immunoaggregation inhibition method (the same applies to latex immunoaggregation method) also changes the molecular size (aggregates) depending on the amount of antigen to be measured. The reaction rate tends to be low and the reaction rate tends to be slow. If the viscosity is high, the adverse effect is noticeable. In addition, since the change in molecular size is observed, the anti-HbA1c antibody immobilized on the supported latex beads must be redispersed as single particles with dissolution after introduction of the diluent. In response to such a requirement, Patent Document 1 provides a reaction cassette having an optimal configuration and function. (1) injection means for introducing a liquid sample into the reaction cassette; (2) in an open liquid flow relationship with the injection means; (i) interacting with the analyte and a function of the analyte And (ii) a reaction having means for perturbing the flow of the liquid by gravity along the reaction path sufficient to contact and stir the liquid And (3) a substantially horizontal axis of rotation, by rotating the reactor about the horizontal axis, the liquid treated in the reaction path moves by gravity along the reaction path An analytical reaction cassette for performing an analytical reaction and measuring an analyte in a liquid sample is provided, characterized in that it comprises a rotating shaft capable of contacting the reagent zone and the flow disturbing means. Yes. The point of Patent Document 1 is that the reagent can be mixed effectively by rotating the provided reaction cassette around a horizontal axis and transferring the fluid by gravity. Using this, a reaction cassette dedicated to urinary microalbumin is also measured by the same measurement principle. However, the DCA2000 system requires a rotation mechanism, a vibration mechanism, and the like in a measuring instrument that is compatible with the reaction cassette partially shown in Patent Document 1, and the apparatus itself becomes larger accordingly. In addition, although depending on the reaction principle in the reaction cassette, since the diluent is sealed in the reaction cassette, there is a limit to the problem of further miniaturization of the reaction cassette.

  On the other hand, Patent Document 2 is a method for detecting or measuring the presence of a biologically specific reactive substance by biologically specific agglutination reaction on carrier particles, wherein the carrier particles form a pearl chain. The method is characterized in that an alternating voltage is applied to the reaction system.

According to Patent Document 2, by applying an alternating electric field to the latex immunoaggregation reaction system, latex particles are arranged linearly along the electric field (pearl chain), and the aggregation reaction is performed by bringing each particle close to each other. I am letting. After that, when the AC electric field is interrupted, the particles are released from the pearl chain, and the particles are re-dispersed by Brownian motion, etc. Since the particles are bonded, the particles do not redisperse even after the AC electric field is interrupted. This method has a higher probability of encountering particles associated with a pearl chain generated by being controlled by an alternating electric field than the probability of encountering particles associated with natural diffusion, and greatly contributes to a reduction in measurement time. In addition, since the size of the particles required for pearl chain formation is several μm, the amount of antibody in the reaction system is small, and the amount of change in size from one particle can be image-analyzed with a microscope or the like. Therefore, it can be measured with high sensitivity as an accompanying effect. From another point of view, according to Patent Document 2, there is a possibility that measurement can be performed without diluting whole blood or plasma by forming a pearl chain of latex particles even in a state including a salt concentration of a physiological saline level. Thus, a liquid reagent-free assay method with good operability can be provided. Furthermore, since it is liquid reagent-free, it is not necessary to enclose a diluting solution or the like when a device such as a reaction cassette as shown in Patent Document 1, so it is an optimal method for miniaturization and chip formation. I think.
Japanese Patent Laid-Open No. 3-46566 Japanese Patent Laid-Open No. 7-83928

  However, when actually measuring a sample such as plasma in a liquid-free manner based on the assay method of Patent Document 2, there is a problem that the redispersibility after the antigen-antibody reaction subsequent to the pearl chain formation is lowered. Since the plasma contains several g / dl (about 5 to 7 g / dl) of protein, it is in a state of higher viscosity than that in a normal buffer solution. In Patent Document 2, it is preferable in that it can form a pearl chain in a form that is not affected by the viscosity to cause particle “meeting” and promote an antigen-antibody reaction, but re-dispersion after the AC electric field is cut off. Because sex is due to diffusion, it is greatly affected by viscosity. If the redispersibility is not good, the pearl chain formation is maintained without being eliminated, so that the state is associated with the antigen-antibody binding force, or the particles do not diffuse due to the influence of viscosity. It is impossible to distinguish whether or not they are lined up next to each other. This is because even if the diffusion over time is tracked, it is difficult to catch the change, and the time required for the diffusion becomes long.

  Therefore, the improvement of the redispersibility after forming the pearl chain has been a problem in the direct measurement of samples such as plasma.

In order to solve the above problems, the measurement chip of the present invention is a measurement chip used for qualitative measurement of a test substance contained in a liquid sample or quantitative measurement of a test substance,
A liquid sample introduction means for introducing the liquid sample;
A liquid sample holding means for holding the liquid sample communicating with the liquid sample introducing means;
An electrode for applying an alternating voltage to the liquid sample introduced into the liquid sample holding means, and a first specific binding to the test substance in a part or all of the liquid sample holding means of the measurement chip A first particle having a volume larger than that of the first particle on which a first particle to which a substance is immobilized and a second specific binding substance that specifically binds to the first specific binding substance to be immobilized on the first particle is immobilized. It is characterized by carrying two particles.

  As a result, since a plurality of particles having different sizes are present, they are easily arranged linearly without being present as aggregates, and dispersibility is improved.

  Specific reaction such as antigen-antibody reaction that can occur after pearl-chained linear particles generated by applying AC voltage by the measuring chip, measuring device, and measuring kit of the present invention, and subsequent interruption of AC voltage It is possible to improve the redispersibility of particles that do not participate in the process. In particular, by using different particles of the present invention in a medium that is highly viscous, such as plasma, where re-dispersibility cannot be obtained by natural diffusion, a difference in diffusion is caused and the effect of re-dispersibility is improved. You can expect As a result, it is possible to provide a measurement chip with very little noise in a device that can measure plasma without diluting it.

  Hereinafter, embodiments of the present invention will be described in detail.

  As an embodiment of the present invention, the measurement chip is used for the presence (qualitative measurement) of the test substance contained in the liquid sample, or for the measurement of the quantity of the test substance (quantitative measurement), The measurement chip includes a liquid sample introduction unit for introducing the liquid sample, a liquid sample holding unit for holding the liquid sample, and a liquid sample holding unit communicating with the liquid sample introduction unit. A first specific binding substance for the test substance is immobilized on a part or all of the liquid sample holding means of the measurement chip and an electrode for applying an alternating voltage to the liquid sample to be introduced. A first particle and a second particle having a volume larger than that of the first particle on which the second specific binding substance that specifically binds to the first specific binding substance immobilized on the first particle is supported. Features that are It is intended to.

  As an example of use of the present invention, the liquid sample is introduced into the measurement chip from the liquid sample introduction means which is a component of the measurement chip, for example, a capillary structure utilizing capillary action. And the liquid sample holding means communicating with the liquid sample introducing means, for example, a fine flow path forming a continuous structure with the capillary structure, and a chamber structure communicating with the flow path. The liquid sample is temporarily or finally held in a thing.

  At that time, the supported first particles and second particles are introduced and held in the measurement chip in a state suspended in the medium of the liquid sample. Thereafter, the suspended particles are formed into a pearl chain by applying an alternating voltage to an electrode in the region where the liquid sample holding means is provided. Here, when the test substance to be measured is contained in the liquid sample, after the AC voltage is cut off, the pearl chain is not partially eliminated but is retained as an aggregate. On the other hand, when the test substance is not included, the pearl chain is eliminated and redispersed. The presence or amount of the test substance is measured based on the degree of redispersion.

  Here, as an important point in the measurement chip of the present invention, the particles having different volumes, that is, the first particles to which the first specific binding substance for the test substance is immobilized, and the first particles are immobilized. The purpose is to use second particles having a volume larger than that of the first particles on which the second specific binding substance that specifically binds to the first specific binding substance is immobilized.

  In general, it is known that diffusion in a medium is inversely proportional to the viscosity of the medium and the spherical equivalent radius of the volume of the material to be diffused, and proportional to the temperature. In a medium with low viscosity, such as in a buffer solution, sufficient diffusion is easily obtained even with the same particle, but in a medium with higher viscosity than water such as plasma, The particles themselves are difficult to move and it is difficult to distinguish whether they are dispersed. Therefore, the present invention aims to promote a redispersibility in a state where a test substance does not exist in the liquid sample by actively introducing different particles to actively cause a difference in diffusion. It is said.

  First, as described above, when an AC voltage is applied to the electrodes in the measurement chip, different particles form a pearl chain in a state of being randomly mixed. At this time, when the first particle and the second particle are arranged, the first particle and the second specific binding substance are immobilized on the respective particles due to the specific binding relationship between the first particle and the second particle. The second particles are bonded.

  Next, when the AC voltage is cut off and the test substance does not exist between the first particles, the first particles, the binding substance of the first particles and the second particles, Different types of volumes will result, which will cause differences in diffusion and better promote dispersion.

  In addition, when the test substance is present between the first particles, if it is held in a form that binds to the second particles, the difference in the volume of the particles is increased, and further good dispersion is possible. It becomes.

  In some cases, depending on the situation in which the test substance exists between the first particles, for example, when the second particles are 4 μm particles and the first particles are 2 μm particles, the second particles and the first particles It is conceivable that there is no difference in particle size as a result, and the diffusion effect cannot be expected.

  However, this can promote diffusion because the factor of the frictional force with the medium is included due to the difference in the shape of the combination. Alternatively, it can be considered that the same particle does not exist as a result by changing the concentration ratio of the first particle and the second particle.

  For example, using the above example, when the second particles are 4 μm particles and the first particles are 2 μm particles, when six particles form a pearl chain, two of the second particles are included in the six particles. It may be included. This makes it possible to give a significant difference in the size of the particles regardless of the order in which the pearl chain is formed or in any form.

  However, since the second particles in the present invention are not intended to be measured and are intended to cause a difference in diffusion, the concentration ratio needs to be lower than that of the first particles. . That is, when the ratio of the first particle to the second particle is equal to or higher than that, only the first particle bonded to the second particle is generated, and thus it is impossible to measure the test substance. .

  In the measuring chip according to the present invention, it always flows as a state of being held by the liquid sample holding means. Generally, it is similar to the relationship between a microscope slide glass and a cover glass, and the particles flow along the flow of the fluid. It is more difficult to stop the particles unless they are settled by gravity or the like. It is. This phenomenon, in combination with the configuration of the present invention, creates a further diffusion effect. That is, a speed difference is caused in accordance with the diffusion, and further effects are exerted on the diffusion. Alternatively, it may be considered that due to the speed difference, large particles become obstacles to the pearl chain afterimage that flows later, further promoting dispersion.

  Here, the dispersion in the present invention is a state in which it is possible to detect a state in which particles are not in contact with each other. For example, when viewed with a microscope, if the space between particles can be recognized, it can be said that the particles are dispersed.

  As an important factor for forming a pearl chain of the present invention, the AC voltage applied to the electrodes of the measurement chip, the frequency of the AC, and the dielectric constant of particles subjected to the action of an AC electric field between the electrodes of the measurement chip, the dielectric constant of the medium, There are particle size and particle concentration. The AC voltage of the present invention may be applied so that the electric field strength is 5 to 50 V / mm, more preferably 10 to 30 V / mm. The AC frequency may be any frequency as long as it is in the range of 10 kHz to 10 MHz.

  From the viewpoint of the physical properties of the particles, it is better for the formation of the pearl chain that the difference between the dielectric constants of the particles and the medium is larger, and it is sufficient if the composition or material can be selected as such. Since such physiological saline is assumed, the dielectric constant of the medium becomes an important factor because the dielectric constant of the medium is fixed much higher than the conductivity of physiological saline of 15 mS / cm. However, in the present invention, the pearl chain can be sufficiently confirmed even with the latex particles usually used. The size of the particles is preferably higher in the polarizability. For example, in the case of latex particles, 0.5 to 10 μm is preferable. The present invention selects particles with different volumes optimally in this range. The higher the particle concentration, the easier it is formed. For example, 0.01 to 1 solid% is preferable for latex particles.

  The measuring chip of the present invention is made of, for example, a general glass or plastic material. For example, if the measuring chip has a three-layer structure, the substrate, the spacer, and the upper cover are stacked in this order. . The adhesive for bonding may be attached to any of the three layers.

  The pressure-sensitive adhesive may be, for example, an acrylic adhesive or a thermoplastic adhesive. In short, any structure may be used as long as the liquid sample introduced into the measurement chip does not leak. The space for the liquid sample introducing means and the liquid sample holding means of the measuring chip can be produced by cutting out a part of the spacer in the example of the three-layer structure.

  The space volume is defined by the area of the cut-out range and the thickness of the spacer. For example, the liquid sample introduction means has a form utilizing a capillary phenomenon. Further, the flow path through which the liquid sample flows or the region carrying the reagent can be configured in the same way. Since the measurement chip of the present invention measures a test substance in the liquid sample, it is important that the liquid sample is introduced into the measurement chip, and further, hydrophilic treatment is performed on the substrate and the upper cover. It is also possible that the air port is provided or an air port is provided at a predetermined place so that the air can be effectively vented.

  Further, in order to prevent the first particles and the second particles from adsorbing non-specifically to the substrate of the measurement chip, for example, the first particles and the second particles may be blocked with a protein such as BSA. For example, the electrode of the present invention is sputtered with a material such as gold, silver, or chromium. The electrode may be arranged on the substrate in an electrode arrangement in which either a uniform electric field or a non-uniform electric field is generated. From the viewpoint of forming a pearl chain, a parallel electrode that generates a uniform electric field is preferable.

  Further, the gap length between electrodes depends on the applied voltage and the electric field strength required for pearl chain formation, but is better because the applied voltage can be reduced as the gap distance is reduced. As an adjunct effect, it also has a positive effect on the amount of specimens. Regarding the relationship between the electrode and the liquid sample holding means, for example, it may be provided between the electrodes along the electrodes, or provided perpendicular to the electrodes. In the former case, a pearl chain is formed in a direction perpendicular to the liquid flow direction. In the latter case, a pearl chain is formed parallel to the liquid flow direction. The former is preferable from the viewpoint of ease of diffusion.

  Although the example so far has been described with a measurement chip having a three-layer structure, the number of layers is not limited to three. For example, a two-layer structure has a function that requires a substrate or an upper cover. It may be molded as follows. In short, it suffices if the liquid sample can be introduced and held in the measuring chip.

  The method for immobilizing the first specific binding substance and the second specific binding substance on the particles of the present invention is based on a known method, that is, a physical adsorption method or a chemical binding method.

  The former physical adsorption method is a method in which the first specific binding substance, the second specific binding substance, and the particles are adsorbed by a hydrophobic binding force or electrostatic force, and their charge state and hydrophobicity are points. Therefore, setting conditions such as pH and salt concentration are important as production conditions.

  On the other hand, in the latter chemical bonding method, amino acid residues existing in the first specific binding substance and the second specific binding substance are bonded to, for example, an amino group. In this case, the particles used have a carbonyl group on the particles, and the carbonyl group is, for example, 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride (abbreviated as EDC) and sulfosuccin. It is easy to handle by reacting with an imide and succinimidyl active in the amino group. In addition, it is possible to use an aldehyde group or an azide-modified one.

  In any case, any bonding method that generates a chemical covalent bond may be used.

  As the dry reagent of the present invention, for example, the prepared particle reagent suspension is spotted on the substrate as it is, and freeze-drying, natural drying, vacuum drying or the like can be considered. Alternatively, lyophilization, natural drying, vacuum drying, or the like may be performed after infiltrating a porous matrix such as filter paper. In any case, the important quality requirements for preparing a dry reagent are the solubility of the dry reagent and the dispersibility of the particles when the dry reagent touches the liquid sample, and storage as a measurement chip. It is sex.

  Taking these into account, the drying process should be established. Here, as the composition of the particle reagent suspension, saccharides such as sucrose and trehalose are further added for the purpose of considering the quality. This is effective as an excipient or preservative for dry reagents.

  Alternatively, a small amount of a surfactant, particularly a nonionic surfactant, may be added for the purpose of improving dispersibility. Or, when pursuing hardness as a dry reagent, for example, a protein such as BSA or a water-soluble polymer may be added. For example, in the case of a three-layer structure, the process of sealing the dry reagent into the measurement chip of the present invention is performed at a stage where the substrate and the spacer are bonded together, and the reagent is placed at a predetermined position of the spacer cutout portion formed on the substrate. After spotting and drying, the upper cover is attached and sealed.

  On the other hand, as described above, in order to produce a stable dry reagent, the excipients and preservatives described above are required, and the measurement system with the measurement chip has a higher viscosity, which makes it possible to disperse after formation of the pearl chain. Is disadvantageous. However, it is considered that this influence can be avoided by adopting the configuration of the present invention.

  In the present invention, as another example of the measurement chip, there may be a case where blood cell separation means is attached to the liquid sample introduction means. Here, the blood cell separation means is, for example, a region where blood cells can be removed along with the rotation of the measurement chip, or a region accompanied by a blood cell filter for blood cell filtration. In any case, by providing the blood cell separating means on the measurement chip, it is possible to directly introduce whole blood obtained by, for example, fingertip blood collection, and to provide a one-step measurement chip.

  The first specific binding substance in the present invention is an antibody against a test substance, and the second specific substance of the present invention has the test substance that specifically binds to the antibody or an antigenic determinant of the test substance. It is a substance. Thereby, the second particles for making a difference in diffusion of the first particles to be measured can be combined, and the second particles have a function like a so-called “bare stone”.

  As another reagent-carrying form of the present invention, the first particle having the first specific binding substance immobilized thereon and the second particle having a volume larger than that of the first particle having the second specific binding substance immobilized thereon are measured. In some cases, the chip is integrally supported. As a result, the reagent carrying steps of the measuring chip can be integrated into one, which is considered to be useful for simplifying the manufacturing process.

  Furthermore, when measuring a low molecular weight compound such as a hapten or a small protein having only one plateau determinant with the measurement chip of the present invention, only a large number of binding sites of the test substance are bound. This can be handled by further supporting the pseudo multivalent binding reagent. This assay chip is used for immunoaggregation inhibition in terms of the assay principle based on antigen-antibody reaction. As the hapten concentration increases, the binding site of the antibody on the first particle is blocked. It is an assay method characterized in that it does not react with a pseudo-multivalent antigen and the aggregation responsiveness decreases.

  For detection of the reaction occurring in the measurement chip of the present invention, a light transmitting means may be provided in the liquid sample holding means area of the measurement chip electrode area. That is, a transparent substrate and upper cover may be used. Thereby, the amount of the aggregate of particles generated according to the amount of the test substance in the measurement chip can be acquired as image data with a microscope or the like, and is quantified by analyzing the image data. be able to.

  As a form of the measuring chip dedicated apparatus of the present invention, at least a measuring apparatus having means for applying and cutting off an alternating electric field to the measuring chip, and means for measuring the particle size distribution change of the particles generated in the measuring chip. If it is. Here, as the particle size distribution measuring means, a microscope lens and a CCD camera or the like are used, acquired as image data, and digitized / quantified by the image analyzing means.

Another embodiment of the present invention is a measurement kit used for measuring the presence of a test substance contained in a liquid sample or the amount of a test substance,
at least,
1) Liquid sample introduction means for introducing the liquid sample;
A liquid sample holding means for holding the liquid sample in communication with the liquid sample introducing means;
An electrode chip having an electrode for applying an alternating voltage to the liquid sample introduced into the liquid sample holding means;
2) a first particle reagent in which a first specific binding substance to a test substance is immobilized,
3) The second specific binding substance that specifically binds to the first specific binding substance immobilized on the first particle comprises a second particle reagent having a volume larger than that of the first particle on which the first particle is immobilized. Provided as a measurement kit.

  In this case, the electrode chip is in a state in which the dry reagent is removed from the measurement chip described in the embodiment. For example, the reagent may be in the form of a liquid sample contained in a container such as an Eppendorf tube or contained in the kit as a dry reagent in a container such as an Eppendorf tube.

The measurement kit of the present invention may further include a jig for separating blood cells.
For example, a centrifuge tube.

  The electrode chip in the measurement kit of the present invention has a light transmissive means. That is, a transparent substrate and upper cover may be used. Thereby, the amount of the aggregate of particles generated according to the amount of the test substance in the electrode tip can be acquired as image data with, for example, a microscope, and is quantified by analyzing the image data. be able to.

  For the dried particles of the first particle reagent and the second particle reagent contained in the measurement kit of the present invention, the idea for the dried reagent is only the difference between whether the electrode chip and the dried body are integrated or not. The measurement chip may be considered to be the same as the dry reagent body enclosed in the measurement chip.

  In the measurement kit of the present invention, the first specific binding substance is an antibody against the test substance, and the second specific substance specifically binds to the antibody, or the substance having an antigenic determinant of the test substance It is. Thereby, the second particles for making a difference in diffusion of the first particles to be measured can be combined, and the second particles have a function like a so-called “bare stone”.

  Furthermore, when measuring a low molecular weight compound such as a hapten or a small protein having only one plateau determinant with the measurement kit of the present invention, for example, a large number of binding sites of the test substance are bound. This can be handled by further supporting the pseudo multivalent binding reagent. This assay chip is used for immunoaggregation inhibition in terms of the assay principle based on antigen-antibody reaction. As the hapten concentration increases, the binding site of the antibody on the first particle is blocked. It is an assay method characterized in that it does not react with a pseudo-multivalent antigen and the aggregation responsiveness decreases.

  As an example of the measurement method of the present invention, a first particle reagent in which a first specific binding substance to a test substance is immobilized and a first specific binding substance immobilized on the first particle are specifically bound. A second particle reagent having a volume larger than that of the first particle on which the second specific binding substance is immobilized and a liquid sample containing the test substance are introduced between the electrode pairs, and application and interruption of the alternating electric field are sequentially performed. This is a method for measuring the presence of the test substance or the amount of the test substance in the liquid sample by measuring the change in the particle size distribution of the particles generated in the electrode pair.

  As an example of the method for measuring the amount of the test substance of the present invention, 1) a step of collecting blood, 2) a step of separating blood cells and plasma using centrifugation or a blood cell filter or the like, and 3) separation 4) The step of taking out only the plasma 4) The first particles on which the second specific binding substance that specifically binds to the first specific binding substance immobilized on the first particles is immobilized using the removed plasma. A step of contacting and mixing with a dry reagent or a liquid reagent containing a second particle reagent having a larger volume, 4) a step of introducing a plasma liquid reagent mixed with the reagent between the electrode pairs, and 4) alternating current with the electrode pairs. A step of applying an electric field, and then shutting off the alternating electric field after a predetermined time, 5) a step of measuring the particle size distribution change of the particles generated in the electrode pair, and 6) using a calibration curve set in advance from the measurement result Liquid The presence of the analyte in the postal or measuring the amount of analyte.

  EXAMPLES Hereinafter, although an Example is shown concretely and this invention is demonstrated in detail, these do not limit this invention.

Example 1 Measurement Chip <Configuration>
A specific measurement chip for carrying out the present invention will be described with reference to FIGS. 2 is a view of the measurement chip 10 of FIG. 1 as viewed from above 11, and FIG. 3 is a view showing a cross section 12 of the measurement chip of FIG. The measurement chip 10 has a three-layer structure of an upper cover 13 made of PET, a spacer 14 which is a double-sided adhesive sheet, and a substrate 15 made of polycarbonate, on which a parallel electrode 16 is formed. The PET upper cover 13 is provided with an injection hole 17 for introducing a liquid sample, and communicates with a space 18 in which the liquid sample is held inside the measurement chip. In the space, the first dry reagent 21 and the second dry reagent 22 are carried to show the diffusion effect of the present invention. In this example, the second dry reagent 22 is a reagent containing 3.56 μm latex beads on which CRP is immobilized, and the first dry reagent 21 is immobilized on an antibody against CRP and an anti-CRP antibody 2 A reagent containing 0.05 μm latex beads. The interelectrode distance 24 was 0.5 mm, and the space width 25 was 1 mm.

  The space thickness 31 is 10 μm from the spacer of the core material 10 μm.

<Production>
Next, a method for manufacturing a measurement chip will be described with reference to FIGS. First, necessary processing is performed in each layer of the measurement chip 10 having a three-layer structure. The upper cover 41 is prepared by cutting the injection hole 17 with a cutting plotter. The spacer 43 is provided with a cut 44 that becomes the space 18 of the measurement chip and a cut 45 that becomes the terminal 23 of the measurement chip. An electrode 47 is formed on the substrate 46 by sputtering. Here, FIG. 4 shows an example in which six measurement chips are manufactured at a time. Here, first, the upper cover 41 and the spacer 43 are bonded together, and the first dry reagent and the second dry reagent are spotted as liquids at predetermined positions in the space 18, and then freeze-dried. What was obtained in this way was bonded to the substrate 46, and finally, the six measurement chips obtained were cut out to obtain the measurement chip 40.

  Here, a method for immobilizing CRP and anti-CRP antibodies on latex beads will be described.

  Immobilization of biological material on latex beads is known as a known technique, and in this example, immobilization was performed by a chemical bonding method. That is, EDC and N-hydroxysulfosuccinimide are reacted with latex beads having a modified carbonyl group to succinimidylate the carbonyl group of the latex beads.

  This is an active group for an amino group that is an amino acid residue of a protein. The activated latex beads thus obtained were immobilized by reacting CRP and an anti-CRP antibody. The form is shown in FIG. The dried first reagent 21 of the measurement chip 10 includes 2.06 μm latex beads 63 on which an anti-CRP antibody 64 is immobilized. On the other hand, the second reagent dry body 22 includes 3.56 μm latex beads 61 to which CRP 62 is immobilized. These specifically bind to each other in the form of a complex 65.

<Measurement and evaluation>
Next, how to use the measuring chip 10 will be described. Here, a measuring device that acts on the measuring chip 10 will be described with reference to FIG. FIG. 5 is a block diagram. FIG. 5 includes an AC voltage control mechanism 51, a lens 52, a CCD camera 53, and an image analysis mechanism 54. In actual use, after plasma is introduced from the injection hole 17 of the measurement chip 10, the terminal 23 of the measurement chip 10 and the AC voltage control mechanism 51 are connected. Thereafter, an AC electric field was generated in the space 18 through the electrode 16 of the measuring chip 10 from the AC voltage control mechanism 51, and after a predetermined time, the AC electric field was cut off, and the particle diffusion state was evaluated from the detection region 55.

  7 and 8 show examples of the diffusion state. FIG. 7 shows the diffusion of one pearl chain. When an alternating electric field is applied, a pearl chain 71 is formed, and then, after a predetermined time, when the alternating electric field is interrupted, the 3.56 μm latex beads 61 function like a cobblestone, causing a difference in diffusion. Thereby, dispersibility is promoted as in 72. Here, what is regarded as an aggregate is one in which 2.06 μm latex beads 62 are continuously connected and held, and the test substance is measured by the ratio of this number to the amount of 2.06 μm latex beads 62.

  Actually, as shown in Fig. 8, the pearl chain of various patterns generated by the AC electric field can make a difference in the diffusion of one pearl chain as shown in Fig. 7 and the encounter with the second pearl chain. However, it further promotes diffusion. Incidentally, it can be said that the function of the plasma fluid in the measuring chip 10 is enhanced by the small laminar flow.

  Here, a measurement chip for dealing with whole blood will be briefly described. As shown in FIG. 9, by inserting a blood cell filter 91 into the injection hole 17 of the measurement chip 10, it is possible to provide a measurement chip 90 that can handle whole blood. That is, when whole blood is spotted from the injection hole 17, blood cells are trapped in the blood cell filter. The plasma passes through the blood cell filter and enters the space of the chip by capillary action. Subsequent mechanisms and actions are the same as described above.

Example 2 Configuration of Measurement Kit FIG. 10 shows a specific measurement kit. The only difference is that the first dry reagent 21 and the second dry reagent 22 carried on the measurement chip 10 are separately put in an eppendle tube or the like. The same.

  According to FIG. 10, the measurement kit 100 includes a centrifuge tube 102 for performing blood cell separation, a tube 103 containing a first dry reagent 21, a tube 104 containing a second dry reagent 22, and an electrode chip 101.

  In use, first, blood is put into the centrifuge tube 102, blood cells are separated by a centrifuge, and plasma is taken out. A predetermined amount of this plasma is taken and sequentially transferred to the tube 102 and the tube 103.

  Thereafter, a liquid sample is introduced into the electrode chip 101 from the tube 103, and thereafter, as shown in Example 1, it is installed in the apparatus of FIG.

  The measurement chip, measurement apparatus, and measurement method of the present invention are a liquid reagent-free high-sensitivity rapid measurement system. Therefore, it is useful as a measurement system used in the POCT field requiring good operability and quick and simple measurement.

The figure which shows the measuring chip used in Example 1 of this invention The figure seen from the direction above the measuring chip used in Example 1 of the present invention Sectional view of the measuring chip used in Example 1 of the present invention The figure which shows the manufacturing process of the measurement chip | tip used in Example 1 of this invention. The figure which shows the block diagram of the measuring apparatus used in Example 1 of this invention Conceptual diagram of reagents used in Example 1 of the present invention Conceptual diagram of diffusion that occurs in Embodiment 1 of the present invention Conceptual diagram of diffusion that occurs in Embodiment 1 of the present invention The figure which shows the measuring chip with the blood cell filter in the measuring chip of this invention The figure which shows the contents of the measurement kit used in Example 2 of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Direction on measurement chip | tip 11,41 12,44 Cross section 13,46 Upper cover 14 Spacer 15 Board | substrate 16 Electrode 17,42 Injection hole 18 Measurement chip inner space 21 1st dry reagent 22 2nd dry reagent 23 Electrode terminal 24 Electrode gap Width 25 Space width 31 Space thickness 44, 45 Cutout part 50 Measuring device 51 AC voltage control mechanism 52 Lens 53 CCD camera 54 Image analysis mechanism 55 Detection region 61, 63 Latex beads 64 Anti-CRP antibody 62 CRP
71, 81 Pearl chain 72, 82 State after dispersion 90 Whole blood measurement chip 91 Blood cell filter 100 Measurement kit 101 Electrode chip 102 Centrifugal tube 103, 104 tube

Claims (14)

A measurement chip used for qualitative measurement of a test substance contained in a liquid sample or quantitative measurement of a test substance,
A liquid sample introduction means for introducing the liquid sample;
A liquid sample holding means for holding the liquid sample communicating with the liquid sample introducing means;
An electrode for applying an alternating voltage to the liquid sample introduced into the liquid sample holding means, and a first specific binding to the test substance in a part or all of the liquid sample holding means of the measurement chip A first particle having a volume larger than that of the first particle on which a first particle to which a substance is immobilized and a second specific binding substance that specifically binds to the first specific binding substance to be immobilized on the first particle is immobilized. A measuring chip which carries two particles. 2. The measuring chip according to claim 1, wherein the liquid sample introducing means comprises a blood cell separating means. The first specific binding substance is an antibody against a test substance, and the second specific substance is the test substance that specifically binds to the antibody, or a substance having an antigenic determinant of the test substance. The measuring chip according to claim 1 or 2. The first particles having the first specific binding substance immobilized thereon and the second particles having a volume larger than that of the first particles having the second specific binding substance immobilized thereon. 3. A measuring chip in the range described in any one of 3 above. 4. The measuring chip according to claim 3, wherein a pseudo multivalent binding reagent in which a large number of binding sites of a test substance are bound is supported. 6. The measuring chip according to claim 1, further comprising a light transmitting means in the liquid sample holding means area of the measuring chip electrode area. 6. A measuring device used for measuring the presence of a test substance contained in a liquid sample or the amount of a test substance, wherein at least an alternating electric field is applied to and cut off from the measuring chip according to claim 1. And a means for measuring a change in the particle size distribution of the particles generated in the measuring chip. 8. The measuring apparatus according to claim 7, wherein the measurement of the particle size distribution change is detected by an image analysis means. A measurement kit used for qualitative measurement of a test substance contained in a liquid sample or quantitative measurement of a test substance,
at least,
A liquid sample introduction means for introducing the liquid sample;
A liquid sample holding means for holding the liquid sample communicating with the liquid sample introducing means;
An electrode chip having an electrode for applying an alternating voltage to the liquid sample introduced into the liquid sample holding means;
A first particle reagent in which a first specific binding substance to a test substance is immobilized;
A second particle reagent having a volume larger than that of the first particle on which the second specific binding substance that specifically binds to the first specific binding substance immobilized on the first particle is immobilized. kit. The measurement kit according to claim 9, wherein the electrode chip has a light transmitting means. The measurement kit according to claim 9 or 10, wherein at least the first particle reagent and the second particle reagent are each in a dry form. The first specific binding substance is an antibody against a test substance, the second specific substance is the test substance that specifically binds to the antibody, or a substance having an antigenic determinant of the test substance. Item 11. The measurement kit according to any one of Items 8 to 10. The measurement kit according to any one of claims 9 to 11, comprising a pseudo multivalent binding reagent in which only a large number of binding sites of a test substance are bound. A first particle reagent in which a first specific binding substance to a test substance is immobilized, and a second specific binding substance that specifically binds to the first specific binding substance to be immobilized on the first particle are immobilized. The particles generated in the electrode pair by introducing a second particle reagent having a volume larger than that of the first particle and a liquid sample containing the test substance between the electrode pairs, and sequentially applying and blocking an alternating electric field. A measurement method characterized by measuring the presence of the test substance or the amount of the test substance in the liquid sample by measuring a change in particle size distribution.

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