JP2009092514A - Sample liquid assay chip and transfer control method of sample liquid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample liquid assay chip having a simple structure and a high storage stability for a plurality of reagents capable of preferably controlling a transfer of a sample liquid. <P>SOLUTION: The sample liquid assay chip 10 includes an electrically insulative substrate 11, on the surface of which a plurality of electrode pairs are formed, and a cover glued to the substrate 11 through a spacer 12. A tubal sample liquid transfer route 25 is present between the substrate 11 and the cover 13. In a non-electrode area in the transfer route 25 where no electrode is present, there are a first and second reagent area 14a and 14b, and a detection area 15 where physicochemical changes in the sample solution are detected. Electrodes 51-58 include exposed parts in which the electrodes are exposed in the transfer route 25. Between the neighboring 2 electrode pairs, a higher voltage is applied to the downstream electrode pair than to the upstream electrode. The sample solution moves along the exposed part of the downstream electrode pair having an improved wettability. By repeating this operation, the sample solution is transferred stepwise to an air hatch 25b. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sample liquid analysis chip for analyzing a sample liquid, and a sample liquid transfer control method in the sample liquid analysis chip.

  In recent years, various substances can be measured by advances in analysis, analysis, and inspection techniques. In particular, in the clinical laboratory field, measurement principles based on specific reactions such as biochemical reactions, enzyme reactions, and immune reactions have been developed, and substances in body fluids that reflect disease states can be measured.

  Currently, in the field of clinical testing, a clinical test called point of care testing (POCT) is drawing attention. POCT means a simple rapid test performed near a patient, and a specific component in a sample solution is quantified in a short time by adopting a simple measurement principle. POCT mainly employs biochemical reactions such as enzyme reactions and antigen-antibody reactions as the measurement principle. This is because enzymes and antibodies not only have a very high reaction specificity for selectively recognizing the reaction relative substances but also have a high reaction efficiency, so that the analyte components in the sample solution can be measured with high sensitivity.

  POCT is generally performed using a sample solution analysis chip. The sample liquid analysis chip is a minute measurement member that can analyze a sample liquid that is a liquid specimen by adopting an arbitrary measurement principle, and is applied to, for example, a biosensor or a cholesterol sensor. At present, in POCT, various studies are being carried out to realize a test method that simplifies the measurement procedure and shortens the time from when a sample is collected until the test result is obtained. Along with this, the sample liquid analysis chip is required to have functions such as being small and portable and adopting a simple measurement principle to facilitate operation and enable high-precision analysis. . Therefore, various studies are currently being conducted to provide a sample solution analysis chip that realizes these demands.

  Therefore, as a sample liquid analysis chip that enables simple and rapid, for example, an insulating substrate provided with an electrode pair consisting of at least a measurement electrode and a counter electrode by screen printing or the like, and an oxidation-reduction provided on the surface of this electrode pair A biosensor having an enzyme layer containing an enzyme and a hydrophilic polymer and an electron acceptor layer has been proposed (see, for example, Patent Document 1). In this biosensor, the electron acceptor is reduced by dropping a sample solution containing a substrate onto the enzyme layer and reacting the enzyme with the substrate. When the reduced electron acceptor is electrochemically oxidized after the completion of the enzyme reaction, the substrate concentration in the sample solution is obtained based on the measured oxidation current value. This biosensor is useful, for example, as a glucose sensor using glucose as a substrate, but is widely used for quantification of various substrates other than glucose.

  By the way, POCT requires a plurality of reagents in some cases. At this time, conventionally, a plurality of reagents are mixed and stored in advance in the sample solution analysis chip. However, there is a problem that the analysis system is lowered due to a change in the characteristics of the reagents. Therefore, as a sample solution analysis chip that can stably store a plurality of reagents, that is, high storage stability, for example, by separately arranging a reagent containing an enzyme and a surfactant and a reagent containing an electron acceptor, A cholesterol sensor with improved storage stability of each reagent has been proposed (see, for example, Patent Document 2). In addition, a cholesterol sensor or the like in which a reagent containing an enzyme and a surfactant and a reagent containing an electron acceptor are arranged separately is shown (for example, see Patent Document 3).

  However, as in the techniques shown in Patent Documents 2 and 3, if a plurality of reagents are arranged separately, the storage stability of each reagent is maintained, but the plurality of reagents are placed in a minute reaction region. When the reagent and the sample solution are mixed at once, there is a problem that it takes a long time to mix the sample solution and each reagent. Further, when the reagents A and B are arranged in order along the flow of the sample solution, the reagent concentration in the sample solution varies, and it is difficult to obtain a uniform sample solution as a whole. If the sample solution and the reagent are not sufficiently reacted in the reaction region, the analysis accuracy is lowered as a result. Therefore, it is important for the sample solution analysis chip to appropriately control the transfer of the sample solution.

  As a method for controlling the transfer of the sample liquid, a method utilizing capillary action is known. The capillary phenomenon is a phenomenon in which the sample liquid moves by a suction force acting between the inner wall surface of the pipe and the sample liquid in the pipe to which the sample liquid is transferred. For example, Patent Document 3 proposes an analysis chip for an immunochromatographic method that uses a porous water-absorbing substance as a chromatographic medium and transfers a sample solution by capillary action. The immunochromatography method is an analysis method using an antigen-antibody reaction, in which a sample solution is passed through a chromatographic carrier on which an antibody is immobilized, and the antibody and a substance to be detected are bound.

Furthermore, in the method of high-throughput nucleic acid sequencing, a method of controlling the transfer of a sample solution by combining centrifugal force or centrifugal force and capillary action has been proposed. In addition, a method has been proposed in which a fluid is transferred by applying a voltage to an electrode provided in a flow path to reduce the surface tension of the sample liquid with respect to the electrode surface (see, for example, Patent Document 4). Such a method is also called a method using an electrowetting phenomenon. For example, in a chemical assay, a microfluidic structure having two-dimensionally arranged electrodes is used to arbitrarily drop droplets two-dimensionally. A moving mechanism has been proposed (see, for example, Patent Document 5). The method using the electrowetting phenomenon can transfer the sample solution efficiently and effectively while having a simple mechanism. In addition, the electrode used for transferring the sample liquid is convenient because it can also be used as a measurement electrode for detecting the reaction between the sample liquid and the reagent.
JP-A-2-062952 JP 2000-039416 A JP 2006-189317 A Japanese translation of PCT publication No. 2002-534096 JP 2005-510347 A

  As described above, a sample solution analysis chip suitable for POCT is useful because it has a simple structure but has high reagent storage stability and can suitably control the transfer of the sample solution. Needless to say, it is important to have high analytical accuracy capable of detecting the reaction between the sample solution and the reagent with high sensitivity.

  In order to increase the storage stability of the reagent, it is effective to store each reagent in a separate area, such as installing a plurality of reaction areas. In addition, it is desirable to install each reagent separately so that the reagents do not mix with each other. However, even if a plurality of reagents are stored separately, if all the reagents are allowed to flow into one reaction region, the concentration distribution of the reagent in the solution in which the sample solution and the reagent are mixed will vary.

  Therefore, in order to improve the above-described variation, a structure may be adopted in which the sample solution is allowed to flow stepwise into each region where each reagent is stored. However, even if a plurality of reaction regions and detection regions are arranged separately, the transfer of sample liquid from the reaction region to the reaction region or from the reaction region to the detection region may not be controlled. For example, if the sample solution is allowed to stay in the reaction region for a long time, the excess reagent is dissolved in the sample solution, so that the sample solution becomes supersaturated. In such a sample solution, since the concentration of the reagent is not uniform, the measurement error becomes large and the analysis accuracy is remarkably lowered. Therefore, it is desired to establish a method capable of arbitrarily controlling the stop / start of the transfer of the sample liquid as well as simply moving the sample liquid.

However, as a method for transferring the sample liquid, for example, in a method using a capillary phenomenon due to a porous water-absorbing substance as in Patent Document 3, the capillary phenomenon is a spontaneous movement of the sample liquid. It is difficult to control the transfer arbitrarily, such as stopping and then starting again. Therefore, it is impossible to control the sample solution for a time required for the reaction between the sample solution and the reagent, for example, after the sample solution is kept in the reaction region and then transferred to the detection region.
On the other hand, in the method of controlling the transfer of the sample liquid by combining the centrifugal force or the centrifugal force and the capillary phenomenon as in Patent Document 4, a rotation mechanism for obtaining the centrifugal force is required, so that the apparatus is complicated. . In addition, since it is necessary to design the sample liquid analysis chip to have a strength that can withstand centrifugal force, the shape and material of the chip are limited.

  Further, in the method using the electrowetting phenomenon as in Patent Document 5, even when the conductivity is sufficiently ensured by exposing an electrode in the sample liquid transfer path, the sample liquid is transferred. Voltage of about several volts is required. In this case, depending on the components in the sample solution, an oxidation-reduction reaction may occur due to energization of the electrode during transfer, and may not be distinguished from a chemical reaction for detecting the reaction between the sample solution and the reagent. This makes it difficult to apply to a sample solution analysis chip that uses an enzyme as a reagent and detects electron transfer due to an oxidation-reduction reaction between the enzyme and the sample solution. In order to suppress such an oxidation-reduction reaction in the vicinity of the electrode, means for forming a dielectric thin film on the surface of the electrode having a large area for transferring the sample liquid is generally known. However, since the voltage applied to the electrode in this case requires several tens of volts or more, it becomes a large-scale device, and it is difficult to say that it is useful for application to POCT for the purpose of simple measurement. there were.

  Therefore, the present invention has been made to solve the above-described problems, and has a simple structure and high storage stability of a plurality of reagents, and also includes transfer of a sample solution including stopping and moving as necessary. An object of the present invention is to provide a controllable sample liquid analysis chip. A further object of the present invention is to provide a sample liquid transfer control method using the sample liquid analysis chip.

  The present inventors have completed the present invention by focusing on the capillary phenomenon and the electrowetting phenomenon as a method for controlling the installation conditions of the reaction region and the detection region and the transfer of the sample liquid, and combining them. .

That is, the above problem is solved by the sample liquid analysis chip of the present invention.
[1] An electrically insulating substrate, an electrode system including two or more pairs of electrodes formed on the substrate, an electrically insulating cover bonded to the substrate through a spacer, and the substrate A tubular flow path defined by the spacer between the cover and a sample liquid inlet having a sample liquid inlet and an air port provided on the opposite side of the inlet; and on the substrate One or two or more reaction reagent regions in which a reaction reagent layer is disposed, a detection region for detecting a physical or optical change of the sample solution, and the inlet A sample liquid supply unit formed on the substrate, wherein the sample liquid supplied to the sample liquid supply unit is sucked from the inlet into the sample liquid transfer path by capillary action And a sample solution analysis chip comprising The electrode system includes a first electrode pair disposed in the vicinity of the inflow port and a second electrode pair adjacent to the first electrode pair, and constitutes the first electrode pair. A part of the electrode is formed outside the sample liquid transfer path from the inlet, and the second electrode pair is formed closer to the air port side of the sample liquid transfer path than the first electrode pair. Further, when arranging three or more N pairs (N = 3, 4, 5...) Of electrode pairs, the Nth electrode pair is the (N−1) th set. The electrodes constituting the electrode pair are arranged symmetrically with respect to the sample liquid transfer path, and are arranged adjacent to the (N-1) th electrode pair on the air port side in the sample liquid supply path with respect to the electrode pair. And at least a part of the sample liquid is disposed in the sample liquid supply section in the region where the sample liquid is supplied, or in the sample liquid transfer path. And the sample liquid analysis chip not placed on a reagent area.
[2] The downstream end of the electrode constituting the electrode pair on the center side of the sample liquid transfer path protrudes toward the downstream side of the sample liquid transfer path, and is adjacent along the sample liquid transfer path The sample solution analysis chip according to [1], wherein the electrodes to be separated are separated through a groove extending in a direction that forms an acute angle with respect to the transfer direction of the sample solution transfer path.
[3] The reaction reagent layer includes an oxidoreductase and an electron mediator, and the detection region is disposed closer to the air inlet side in the sample liquid transfer path than the reaction reagent region [1] or The sample liquid analysis chip according to [2].
[4] The detection region includes a measurement electrode pair that is disposed on a surface of the substrate and includes a measurement electrode and a counter electrode, and the measurement electrode is disposed in a central portion of the detection region, and the measurement electrode The sample liquid analysis chip according to [3], wherein the width of is narrower than the width of the sample liquid transfer path.
[5] The sample liquid analysis chip according to [4], wherein the measurement electrode is covered with the reaction reagent layer.
[6] The sample liquid analysis chip according to any one of [1] to [5], wherein at least a region that can be the detection region of the substrate or the cover is formed of a light-transmitting material.
[7] In any one of [1] to [6], in the sample solution transfer path, a sensing region for detecting the position of the tip of the sample solution is arranged in the vicinity of a boundary portion between the adjacent electrode pairs. Sample liquid analysis chip.

Moreover, the said subject is solved by the transfer control method of the sample liquid of this invention.
[8] A step of preparing the sample liquid analysis chip according to [1], a step of dropping a sample liquid so as to at least partially overlap the first electrode pair and the second electrode pair, and the sample Applying a voltage higher than the voltage of the second electrode pair to the first electrode pair in which the liquid partially overlaps, and further, three or more N sets (N = 3) 4, 5,...)), The air port side of any two pairs of electrode pairs adjacent to each other along the flow of the sample solution in the sample solution transfer path. The tip of the sample solution in the sample solution transfer path is brought into contact with the electrode pair arranged at the position of the electrode pair, and the electrode pair is arranged on the inlet side of the two arbitrary electrode pairs. Applying a voltage higher than the voltage of the electrode pair arranged on the air port side to the electrode pair. A method for controlling the transfer of the sample liquid.
[9] A step of preparing the sample liquid analysis chip according to [7], a step of dripping a sample liquid so as to at least partially overlap the first electrode pair and the second electrode pair, and the sample The step of applying a voltage higher than the voltage of the second electrode pair to the first electrode pair in which the liquid partially overlaps, and the sample liquid is in contact with one electrode pair in the sensing region In the case where three or more N pairs (N = 3, 4, 5,...) Of electrode pairs are arranged. The tip portion of the sample liquid in the sample liquid transfer path is in contact with the electrode pair arranged on the air port side of any two adjacent electrode pairs along the flow of the sample liquid After detecting that the sample liquid is detected by the sensing area, the sample liquid is placed on the air inlet side. With respect to the electrode pair disposed on the inlet side of the two pairs of electrodes, the voltage of the electrode pair disposed on the air port side with respect to the electrode pair disposed on the inlet side of the two electrode pairs in a state of being in contact with and stationary. A method for controlling the transfer of a sample solution, including a step of applying a higher voltage.
[10] The sample liquid transfer control method according to [9], wherein the electrical change is sensed as a decrease in a resistance value between any two adjacent electrode pairs.
[11] A step of preparing the sample liquid analysis chip according to [7], a step of dripping a sample liquid so as to at least partially overlap the first electrode pair and the second electrode pair, and the sample The step of applying a voltage higher than the voltage of the second electrode pair to the first electrode pair in which the liquid partially overlaps, and the sample liquid is in contact with one electrode pair in the sensing region In the case where three or more N pairs (N = 3, 4, 5,...) Of electrode pairs are arranged in the sample liquid transfer path. Of the two electrode pairs adjacent to each other along the flow of the sample liquid, the tip of the sample liquid in the sample liquid transfer path contacts the electrode pair on the air port side of the sample liquid transfer path After the detection area senses that the sample liquid is placed on the air inlet side. Applying a voltage higher than the voltage of the electrode pair disposed on the air port side to the electrode pair disposed on the inflow port side in a state where the electrode pair is in contact with and stationary. Sample liquid transfer control method.

  According to the present invention, it is possible to provide a sample liquid analysis chip that has a simple structure but has a high storage stability of a plurality of reagents, and can control the transfer of a sample liquid including stopping and moving as necessary. . Further, in the sample liquid analysis chip, the transfer of the sample liquid can be suitably controlled by utilizing the electrowetting phenomenon caused by a plurality of electrode pairs. Therefore, the sample liquid analysis chip and the sample liquid transfer control method of the present invention are useful for simple rapid inspection such as POCT.

[Analysis chip]
FIG. 1 is a diagram showing an example of an embodiment of the sample liquid analysis chip of the present invention. As shown in FIG. 1A, the sample liquid analysis chip 10 includes a substrate 11, a pair of spacers 12, and a cover 13. The sample liquid analysis chip 10 has an integrated structure in which the substrate 11 and the cover 13 are bonded to each other through the spacer 12, and includes a tubular space defined by the spacer 12 between the substrate 11 and the cover 13. This space becomes a sample liquid transfer path which is a flow path of the sample liquid. In FIG. 1A, a broken line portion denoted by reference numeral 25 is a portion that becomes a sample liquid transfer path.

  On the surface of the substrate 11, a first reaction reagent region 14 a, a second reaction reagent region 14 b, and a detection region 15 are arranged in a region inside the sample liquid transfer path 25. Further, an electrode system is formed on the surface of the substrate 11 in a region where these reaction reagent regions are not arranged (not shown in FIG. 1). The electrode system means an assembly of electrode pairs including two or more pairs of electrodes (electrode pairs), and is used for controlling the transfer of the sample liquid. The electrode pair constituting the electrode system, the method for controlling the transfer of the sample solution, etc. will be described in detail later.

  The substrate 11 is formed of an electrically insulating material for the purpose of preventing electric leakage. The electrically insulating material is not particularly limited, and examples thereof include polyethylene terephthalate (PET), polycarbonate, polystyrene, polypropylene, and polyester. The substrate 11 may be in the form of a very thin film or a plate having a certain thickness. The thickness of the substrate 11 is not particularly limited, but it is preferable that the thickness is about 100 to 1000 μm from the viewpoint of realizing downsizing of the sample liquid analysis chip and improving portability. .

  The spacer 12 is disposed to face an arbitrary position on the substrate 11. The width of the sample liquid transfer path 25 is defined by the distance between the facing spacers 12. The material forming the spacer 12 may be the same material as the substrate 11 and is not particularly limited. The spacer 12 acts as an adhesive member that joins the substrate 11 and the cover 13 together. Therefore, the surface of the spacer 12 may have an adhesive layer for improving the adhesion with the substrate 11 and the cover 13. The adhesive layer refers to a layer formed with a general-purpose adhesive or the like. Although the kind of adhesive agent is not specifically limited, What does not react with the board | substrate 11 and the cover 13, and does not melt | dissolve is good. Specific examples include acrylic resins.

  The cover 13 is bonded to the substrate 11 via the spacer 12 to form the sample liquid transfer path 25. The cover 13 is preferably made of an electrically insulating material for the purpose of preventing electric leakage as with the substrate 11. The electrically insulating material is not particularly limited, and examples thereof include polyethylene terephthalate (PET), polycarbonate, polystyrene, polypropylene, and polyester. At this time, it is preferable that at least a region (detection region) for detecting a reaction change between the sample solution and the reagent in the base material 11 and the cover 13 is formed of a material having high light transmittance. Thus, if the light transmittance of the part which comprises a detection area | region among the base material 11 and the cover 13 is high, the sample liquid analysis chip | tip 10 will be able to detect the change of a sample liquid easily optically. The detection area will be described in detail later.

  The first reaction reagent region 14a and the second reaction reagent region 14b are regions where reaction reagent layers are arranged. The reaction reagent layer refers to a solid or semi-solid (gel etc.) layer containing a reagent for advancing the reaction of a specific component in a sample solution. The specific component in the sample solution may mean a substance to be detected that is a main analysis target. The reaction reagent region is appropriately determined according to the number of reagents used. At this time, there may be one or more reaction reagent regions on the substrate 11. When two or more reaction reagent regions are formed, even when a plurality of reagents are required for inspection, the reagents are not mixed and stored in one place until the sample solution and the reagent are reacted. Can be stored separately, so that the storage stability of each reagent is kept high. In addition, since the reaction between the sample solution and the reagent can be performed individually, rather than reacting a plurality of reagents and the sample solution at the same time, the sample solution and the reagent can be reacted uniformly without unevenness, As a result, the analysis accuracy is improved. When one or more reaction reagent layers are installed in the reaction reagent region 14, the interval between the reaction reagent layers is not particularly limited, and may be determined as appropriate according to the shape and dimensions of the sample liquid analysis chip.

  Examples of the main reagent contained in the reaction reagent layer include an enzyme that catalyzes a chemical reaction of a specific component in a sample solution, a substance that specifically binds to the specific component, and the like. Examples of the enzyme include cholesterol oxidase that catalyzes oxidation of cholesterol, cholesterol dehydrogenase, and the like. A reaction system can be constructed using glucose oxidase if the measurement target is glucose, and using lipoprotein lipase and glycerol dehydrogenase if the measurement target is triglyceride (neutral lipid).

A substance that specifically binds to a specific component means a substance that binds chemically or physically. This includes an antibody having a specific binding ability to an antigen. A well-known thing can be used for an antibody. Specific examples include anti-albumin antibodies, anti-HCG antibodies, anti-IgA antibodies, anti-IgM antibodies, anti-IgE antibodies, anti-IgD antibodies, anti-AFP antibodies, anti-DNT antibodies, anti-prostaglandin antibodies, anti-human clotting factor antibodies, anti-human coagulation factor antibodies, CRP antibodies, anti-HBs antibodies, anti-human growth hormone antibodies, antisteroid hormone antibodies and the like are included. Alternatively, particles carrying antibodies may be used. The particles for supporting the antibody may be arbitrarily selected and are not particularly limited. For example, magnetic metal powder, Fe ₃ O ₄ , γ-Fe ₂ 0 ₃ , Co-γ-Fe ₂ 0 ₃ , nylon And fine particles of a polymer such as polyacrylamide and ferrite and the like. These particles can be formed by integrating the desired polymer and ferrite through a silane coupling agent or the like, and can also be formed by chemically bonding to fine particles of a polymer such as polystyrene. Can do.

  Further, the reaction reagent layer may contain an oxidoreductase and an electron mediator. An oxidoreductase is an enzyme that serves as a catalyst for a redox reaction involving electron transfer caused by an electron mediator described later. An electron mediator refers to an electron transfer medium that acts as an oxidizing agent that receives electrons from cholesterol, glucose, and the like, and is in a reduced state. Examples of electron mediators include ferricyanide ions. Ferricyanide ions are generated when potassium ferricyanide is dissolved in an aqueous solution. Ferricyanide ions receive electrons from cholesterol, glucose and the like and become ferrocyanide ions.

  The reaction reagent layer may contain a surfactant for activating the enzyme reaction as necessary. Examples of the surfactant include polyoxyethylene-pt-octylphenyl ether and sodium cholate. However, the wettability of the sample liquid with respect to the inner wall surface of the sample liquid transfer path 25 is improved by contacting the reaction reagent layer containing the surfactant. The sample liquid with improved wettability may spontaneously move downstream in the sample liquid transfer path 25. Therefore, it may be difficult to control the sample liquid transfer well even by the sample liquid transfer control method described later. Therefore, when forming a reaction reagent layer containing a surfactant, it is preferable to dispose the reaction reagent layer on the most downstream side in the sample liquid transfer path 25.

  Further, the reaction reagent layer may contain substances such as taurine, maltitol, carboxymethylcellulose and the like. The reaction reagent layer containing these substances has a uniform surface shape, and when the sample solution comes into contact with the reaction reagent layer, the reagent in the reaction reagent layer can be quickly dissolved in the sample solution.

  The detection region 15 is a region where a change occurring in the sample liquid after reacting with the reagent is detected. Changes in the sample liquid include physical changes in the sample liquid in which the properties of the sample liquid change and chemical changes in which a specific component of the sample liquid is chemically changed to another substance. Here, the change in properties means a change in color, a change in state from liquid to solid, and the like. The change in the sample liquid is detected optically or electrically by a desired detection means. Optical detection of changes in the sample liquid means that the behavior of the chemical or physical change in the sample liquid is determined by measuring the absorbance, transmittance, or solid content in the sample liquid within a certain range of electromagnetic waves of a specific wavelength. Judgment is based on the appearance of the object. Examples of means for optically detecting a change in the sample solution include an absorptiometer and an image processing apparatus. Further, the optical detection means includes observing changes in the sample solution with the naked eye.

  When optically detecting a change in the sample solution, the electromagnetic wave used for detection (visible light in the case of visual observation) needs to pass through the detection region 15. Therefore, the member constituting the detection region 15 is preferably formed of a material having high light transmittance. The material having high light transmittance is a material having high light transmittance including visible light, and may be a thermoplastic resin. Among these, polyethylene terephthalate and polycarbonate are preferable, and polycarbonate is particularly useful because it exhibits very high light transmittance with respect to visible light.

  The electrical detection of the change in the sample solution means that the behavior of the chemical or physical change in the sample solution is determined based on the electrical change. An electrode or the like is formed in the detection region 15 when a change in the sample solution is electrically detected. As an electrode system used in the detection region 15, a measurement electrode pair composed of a measurement electrode and a counter electrode is useful. A measuring electrode pair and a method for electrically detecting a change in the sample solution by the measuring electrode pair will be described later.

  Moreover, it is preferable that the cover 13 of the area | region in which at least the electrode for a measurement was formed in the board | substrate 11 is formed with the light transmissive material. This is because a change in the sample solution caused by the reaction between the sample solution and the reagent can be detected visually.

  FIG. 1B is a cross-sectional view of the integrated sample solution analysis chip 10 cut in the longitudinal direction at the center thereof. As shown in FIG. 1B, the integrated sample solution analysis chip 10 includes a sample solution supply unit A and a sample solution transfer unit B including a sample solution transfer path 25. The sample liquid supply unit A is an area that is located outside the sample liquid transfer path 25 and includes an inlet 25a of the sample liquid transfer path 25 described later. In the inspection, the sample liquid is dropped at an arbitrary position of the sample liquid supply unit A.

  The sample liquid transfer path 25 is a tubular flow path through which the sample liquid flows. An inlet 25 a and an air port 25 b are formed at both ends of the sample liquid transfer path 25. The inflow port 25a is an opening for introducing the sample liquid from the sample supply unit A. On the other hand, the air port 25b is formed at the terminal end opposite to the inflow port 25a, and discharges the air in the sample liquid transfer path 25.

  In order to transfer the sample solution, the wall surface of the sample solution transfer path 25 needs to have wettability with respect to the sample solution or the sample solution in which the reaction reagent is dissolved. However, if the wettability of the wall surface of the sample liquid transfer path 25 is high, the sample liquid moves spontaneously, so that it is difficult to control the transfer of the sample liquid. Therefore, the sample liquid transfer path 25 is formed of a material having low wettability with respect to the sample liquid. For example, when the sample solution is an aqueous solution such as blood, a polymer film such as PET (polyethylene terephthalate) or polycarbonate having a relatively low wettability to the sample solution is useful. Moreover, it is good also as desired wettability by coat | covering at least the surface which a sample solution contacts among the board | substrate 11, the spacer 12, and the cover 13 which comprise the sample solution transfer path 25 with arbitrary materials.

  The sample liquid transfer path 25 preferably has a square cross-sectional shape. The dimension of the cross section of the sample liquid transfer path 25 affects wettability that is important in controlling the transfer of the sample liquid. When transferring the sample liquid, the sample liquid spontaneously flows from the sample liquid supply section A into the sample liquid transfer path 25, or the sample liquid reversely flows when the sample liquid is transferred in the sample liquid transfer path 25. Should be avoided as much as possible. In order to prevent turbulent flow such as inflow and back flow of the sample liquid, when the cross section of the sample liquid transfer path 25 is a square, the width of the cross section is 0.5 mm or more and 5 mm or less, and the height is in the range of 5 to 500 μm. It is preferable that it exists in. By adjusting the cross-sectional shape of the sample liquid transfer path 25 in this way, the wettability of the sample liquid with respect to the sample liquid transfer path 25 can be suitably controlled, so that the turbulent flow of the sample liquid in the sample liquid transfer path 25 can be suppressed. .

  If the reaction reagent region 14 and the detection region 15 are installed separately as in the present embodiment, the sample solution flows due to the stirring effect when the sample solution is transferred from the reaction reagent region 14 to the detection region 15. A uniform sample solution is obtained in the detection region.

  Next, an electrode system necessary for sample liquid transfer control will be described. The electrode system includes two or more pairs of a pair of electrodes formed on the substrate. FIG. 2 is a schematic view of the sample liquid analysis chip shown in FIG. 1 as viewed from above. 2, the illustration of the cover is omitted, and the same reference numerals are used for the same members as those shown in FIG. Hereinafter, the structure of the electrode system will be mainly described, and a method for controlling the transfer of the sample solution using the electrode system will be described in detail later.

  As shown in FIG. 2, a plurality of electrodes 51 to 58 for controlling the transfer of the sample liquid are formed on the substrate 11. The electrodes 51, 53, 55, and 57 are disposed symmetrically with respect to the electrodes 52, 54, 56, and 58 and the sample solution transfer path 25, respectively. One pair of electrodes is composed of the electrodes to be processed. In addition, grooves 71 to 76 are cut between adjacent electrodes along the flow of the sample solution, and the electrodes 51 to 58 are separated from each other. The grooves 71 to 76 define the shape of each electrode important for the transfer of the sample liquid. In order to appropriately transfer the sample liquid, the downstream ends of the electrodes 51 to 58 near the sample liquid transfer path on the center side of the sample liquid transfer path 25 protrude downstream. That is, the grooves 71 to 76 in the vicinity of the sample liquid transfer path 25 are provided so as to form an acute angle with respect to the flow of the sample liquid.

  Thus, four electrode pairs are formed on the substrate 11 of the present embodiment. These electrode pairs are arranged adjacent to each other along the flow of the sample solution. When N pairs (N = 3, 4, 5,...) Of electrode pairs of 3 or more are arranged, the N-th electrode pair is compared to the (N−1) -th electrode pair. Thus, it is only necessary to be formed adjacent to the air port 25b side in the sample liquid transfer path 25. In the following description, the electrode pair arranged on the most upstream side along the flow of the sample liquid is referred to as a first electrode pair, and the second, third, and fourth are arranged in the order of arrangement toward the downstream side of the sample liquid. This is referred to as an electrode pair.

  The first electrode pair includes an electrode 51 and an electrode 52. A part of the electrodes 51 and 52 is disposed on the sample liquid supply part A outside the sample liquid transfer path 25, and the downstream end of the electrodes 51 and 52 on the center side of the sample liquid transfer path 25 is The shape protrudes to the vicinity of the inflow port 25a. The second electrode pair is composed of an electrode 53 and an electrode 54. The electrodes 53 and 54 are arranged adjacent to the air port 25b side of the sample liquid transfer path 25 with respect to the electrodes 51 and 52 constituting the first electrode pair.

  The third electrode pair includes an electrode 55 and an electrode 56. The electrodes 55 and 56 are disposed adjacent to the air port 25b side with respect to the electrodes 53 and 54 constituting the second electrode pair. The fourth electrode pair includes an electrode 57 and an electrode 58. The electrodes 57 and 58 are arranged closest to the air port 25b in the sample liquid transfer path 25.

  The electrodes 51 to 58 constituting the first to fourth electrode pairs are all metal thin films and are separated by grooves 71 to 76 provided between the electrodes. Further, a non-electrode region 59 exists between the electrodes 51, 53, 55, 57 and the electrodes 52, 54, 56, 58. The non-electrode region 59 is a region where each electrode is not disposed and does not function as an electrode. Inside the non-electrode region 59, reaction reagent regions 14a and 14b are installed. In order to transfer the sample liquid, a voltage is applied to one of the first to fourth electrode pairs depending on the region to be transferred. When the electrodes 51 to 58 are activated by energization, the wettability on the surface is improved, so that the sample solution spreads over the surface of the energized electrode due to the electrowetting phenomenon. At this time, since no voltage is applied to the reaction reagent regions 14a and 14b provided in the non-electrode region 59, the characteristics of the reaction reagent layer are not changed by energization.

  FIG. 3 is an enlarged view of the vicinity of the inlet 25a. As shown in FIG. 3, parts of the electrodes 53 to 56 are exposed in the sample liquid transfer path 25. This exposed portion is also referred to as an “exposed portion”. The exposed portion refers to a portion of the electrode that is exposed to the sample solution flow path in the sample solution transfer path 25 and contacts the sample solution. When a voltage is applied to the pair of electrodes including the exposed portion, the exposed portion is activated and the hydrophilicity is increased. When the hydrophilicity of the exposed portion is increased, the wettability with respect to the sample liquid is also improved. At this time, the sample liquid that has been stationary on the upstream side of the exposed portion with increased hydrophilicity moves so as to be drawn to the exposed portion with increased hydrophilicity. The fourth electrode pair omitted in FIG. 3 also has a similar exposed portion.

  The behavior related to the movement of the sample solution depends on the width x1 of the exposed portion and the shape of the exposed portion. The width x1 of the exposed portion is appropriately set according to the width X of the sample liquid transfer path 25. The width x1 of the exposed portion refers to the shortest distance from the wall surface of the sample solution transfer path 25 to one side that constitutes a substantially parallel electrode along the flow of the sample solution in the exposed portion. It is preferable that x1 / X, which is a ratio between the length x1 of the exposed portion and the width X of the sample liquid transfer path 25, is 1/20 or more and 1/4 or less.

  For example, when x1 / X is 1/4, half of the entire width of the sample liquid transfer path 25 becomes an exposed portion that functions as an electrode, and half of the remaining width excluding the exposed portion does not function as an electrode. It becomes a region (non-electrode region). Thereby, it is possible to secure a sufficient area for arranging the reaction reagent layer and the electric power necessary for transferring the sample liquid. However, when x1 / X exceeds 1/4, the non-electrode region of the sample liquid transfer path 25 becomes very narrow. Therefore, when a voltage is applied to the electrode pair, the sample liquid flowing in the sample liquid transfer path 25 is energized. There is a fear. When the sample solution is energized, the sample solution is electrochemically denatured and the detection accuracy is reduced. In addition, it becomes difficult to control the transfer of the sample solution, and in some cases, the sample solution is mixed into the detection region, so that the reaction reagent in which the electron mediator is dissolved is reduced by the interaction with the electrode regardless of the measurement target. Since the reduction reaction of such a reaction reagent is difficult to distinguish from the reduction reaction by an enzyme, it is difficult to accurately measure the measurement target substance. On the other hand, when x1 / X is less than 1/20, the exposed portion becomes excessively small, so it is difficult to improve the wettability of the wall surface of the sample liquid transfer path 25.

  In order to more reliably transfer the sample liquid and suppress damage to the sample liquid due to voltage, x1 / X is more preferably 1/10 or more and 1/5 or less. For example, when the width X of the sample liquid transfer path 25 is 1.0 mm, the width x1 of the exposed portion may be set to 0.1 mm to 0.2 mm or less.

  The width x2 of the region that does not function as an electrode while being sandwiched between the electrode pairs is preferably such that x2 / X, which is a ratio to the width X of the sample liquid transfer path 25, is 1/2 or more and 9/10 or less. In addition, the length L of one side of the exposed portion, which is on the central portion side of the sample solution transfer path 25 and extends in the direction of transferring the sample solution, is at least larger than the diameter D of the reaction reagent layer. More preferably, the length L of one side is 2 or more with respect to the diameter D of the reaction reagent layer. Thereby, an electrowetting phenomenon sufficient for the transfer of the sample liquid can be caused.

  The shape of the exposed portion is designed so that the sample solution can be appropriately moved from the upstream side to the downstream side. The exposed portion of the electrode is arranged so that the downstream end portion on the center side of the sample liquid transfer path 25 protrudes toward the downstream side of the sample liquid transfer path 25. When a voltage is applied to an electrode installed in the vicinity of the sample solution, the sample solution starts to move along the shape of the exposed portion whose wettability is improved by energization. Therefore, if the shape of the exposed portion is defined as described above, the sample liquid is reliably guided from the upstream side to the downstream side by the exposed portion activated by energization. The behavior of the sample solution is controlled by the energized exposed portion and is not affected by the unexposed exposed portion. For this reason, the sample solution is stationary in the vicinity of the boundary between the energized exposed portion and the unexcited exposed portion.

  Next, the sample liquid transfer control method of the present invention will be described using the sample liquid analysis chip 10 shown in FIG. In the present invention, when the sample liquid is transferred to the sample liquid transfer path 25, a capillary phenomenon due to the wettability of the sample liquid to the wall surface in the sample liquid transfer path is used.

The relationship of the following equation is known between the force at which the liquid tries to enter the capillary and the height of the capillary rise when the capillary is inserted vertically into the liquid (Kazuo Tajima et al., “Interface Chemistry”). ", P41, Maruzen, 2005).

  From the above formula, if the contact angle between the liquid and the capillary surface is 90 degrees or more, the right side is zero or negative, and the capillary rising height h is also zero or negative. That is, no liquid can enter the capillary. Therefore, it can be seen that the capillary rise does not occur unless the contact angle between the liquid and the capillary surface is 90 degrees or less.

  When transferring a sample solution by capillary action, the wettability of the wall surface of the sample solution transfer path is important. Therefore, in the present invention, as described above, an exposed portion (a portion exposed in the sample liquid transfer path) is provided on the electrodes constituting the electrode pair to control the wettability of the electrode in contact with the sample liquid. At this time, the sample solution is transferred stepwise from the upstream side to the downstream side by performing an appropriate energization operation in order from the upstream electrode pair to the downstream electrode pair along the flow of the sample solution. .

  Therefore, the sample liquid transfer control method of the present invention includes (1) a step of preparing a sample liquid analysis chip according to the present invention as described above, and (2) a first electrode pair and a second electrode pair. A step of dropping the sample solution so as to at least partially overlap; and (3) a step of applying a voltage higher than the voltage of the second electrode pair to the first electrode pair to which the sample solution has been dropped. And having. Further, when three or more sets of N pairs (N = 3, 4, 5,...) Of electrode pairs are arranged, any two adjacent along the flow of the sample solution in the sample solution transfer path. Of the pair of electrodes, the two pairs of electrodes are arranged in a state where the tip portion of the sample solution in the sample solution transfer path is in contact with the electrode pair arranged on the air port side and is stationary. A voltage higher than the voltage of the electrode pair arranged on the air port side is applied to the electrode pair arranged on the inlet side among the pair. This step may be repeated.

  In the step (1), the sample liquid analysis chip according to the present invention as described above is prepared. The members constituting the sample liquid analysis chip are the same as described above, and thus the description thereof is omitted. The method for manufacturing the sample liquid analysis chip will be described in detail later.

  In the step (2), the sample liquid is dropped to introduce the sample liquid into the sample liquid analysis chip 10. It is preferable that the sample liquid is dropped so as to at least partially overlap the first electrode pair and the second electrode pair. More preferably, it is dropped before the inflow port 25a and in contact with the inflow port 25a.

  The sample liquid analysis chip 10 may have a spotting part. The spotting part is a part where the sample liquid is dropped so as to facilitate the inflow of the sample liquid into the sample liquid transfer path 25 in the sample supply part A. The spotting part may include a layer containing a substance that facilitates the movement of the sample liquid. The spotting unit is preferably installed on the upstream side of the inflow port 25a, but the installation site is not particularly limited as long as the installation site is inside the sample supply unit A. In addition, when the sample solution is blood, it may be necessary to remove solid components such as blood cells. In this case, a filter for removing blood cells in the blood may be installed at an arbitrary location on the substrate 11. The installation location of the filter is not particularly limited, but it is preferably installed in the sample solution transfer path in order to remove the solid component in the sample solution.

  In the step (3), first, a voltage higher than the voltage applied to the second electrode pair is applied to the first electrode pair to which the sample solution has been dropped. The voltage applied to the second electrode pair is preferably a positive voltage. At this time, the sample solution is guided from the sample solution supply unit A into the sample solution transfer path 25 along the first electrode pair activated by energization. Furthermore, the sample solution moves downstream along the exposed portion of the activated second electrode pair in the sample solution transfer path 25. By the movement, the sample solution comes into contact with the first reaction reagent region 14a sandwiched between the second electrode pair, and the reagent is gradually dissolved in the sample solution. Thereby, the reagent such as an enzyme held in the first reaction reagent region 14a reacts with the sample liquid. The sample liquid guided into the sample liquid transfer path 25 is adjacent to the downstream side of the second electrode pair and comes into contact with an inactive third electrode pair that is not yet energized and stops still.

  After the reaction between the reagent held in the first reaction reagent region 14a and the sample liquid is completed, a voltage higher than the voltage of the third electrode pair is applied to the second electrode pair. As before, the sample solution starts to spread so as to cover the exposed portion of the third electrode pair activated and improved in wettability, and is transferred to the downstream side of the third electrode pair. The movement of the sample solution proceeds to the boundary between the third electrode pair and the fourth electrode pair, but comes into contact with the inactive fourth electrode pair that is not yet energized and stops.

  At this time, the sample solution is guided into the second reaction reagent layer 14b sandwiched between the third electrode pair, and the reagent stored in the second reaction reagent region 14b is gradually dissolved in the sample solution. . Since the reagent in the first reaction reagent region 14a is already dissolved in the sample solution that has reached the second reaction reagent region 14b, the reaction between the new reagent and the sample solution is suitably performed. As described above, in the present invention, a plurality of reactions can be advanced step by step without excess and deficiency, and the sample solution in which the reagent is uniformly dissolved can be transferred to a new reaction reagent layer. Moreover, since the turbulent flow of the sample liquid can be suppressed by suitably adjusting the dimension of the sample liquid transfer path 25 within a predetermined range, the sample liquid can be reliably transferred to the reaction reagent layer.

  After the reaction between the sample solution and the reagent is completed in the second reaction reagent region 14b, a voltage higher than the voltage of the fourth electrode pair is applied to the third electrode pair. Along with this, the sample liquid is further guided to the air port 25 b side in the sample liquid transfer path 25. Since the fourth electrode pair extends to the vicinity of the air port 25b, the sample liquid is guided to the air port 25b and stops. In addition, the area | region pinched | interposed by the 4th electrode pair on the surface of the board | substrate 11 does not have a metal thin film coating | cover, and the transparent insulating board | substrate 11 is exposed. Here, since the cover 13 is formed of a highly light-transmitting material, it is possible to optically detect a change in the sample liquid using the region sandwiched between the fourth electrode pairs as a detection region. . This change in the sample solution includes a change in which components of the first reaction reagent region 14a and the second reaction reagent region 14b are dissolved in the sample solution and a predetermined chemical reaction occurs.

  Further, when a direct voltage is applied to the electrode pair, the electrode pair on the lower potential side has a greater effect of improving the wettability of the droplets on the electrode surface. Therefore, when a voltage is applied between adjacent electrode pairs, it is preferable to apply a voltage so that the electrode pair on the air port side 24 becomes positive.

  As described above, in the present invention, a plurality of electrode pairs are arranged adjacent to each other along the flow of the sample solution, and voltage is applied to two pairs of electrode pairs arbitrarily selected from them. Any two pairs of electrodes may be selected from the electrode pair A on the inlet side and the electrode pair B on the air port 25b side that are close to the tip of the sample solution. Here, the voltage of the electrode pair A on the upstream side with respect to the direction in which the sample liquid is transferred is preferably a positive voltage higher than the voltage applied to the electrode pair B, more preferably the voltage applied to the electrode pair A. Is set to +2 to 3 V higher than the voltage applied to the electrode pair B. Thereby, the wettability of the electrode pairs A and B activated by energization is improved, and the sample liquid starts to spread so as to cover the surface of the electrode pair B and is gradually transferred to the air inlet side. Here, if the voltage applied to the electrode pair A is lower than the voltage applied to the electrode pair B, the sample solution can be transferred. However, the voltage applied to the electrode pair A is set higher than the voltage applied to the electrode pair B as in the present invention. As compared with the case where it is also increased, the transfer speed is clearly reduced.

  Thereafter, the sample liquid moves downstream over the surface of the electrode pair B and stops at the vicinity of the downstream outer side of the electrode pair B. This is because the downstream outer side of the electrode pair B that is not energized is inactive, and the wettability necessary for transferring the sample liquid is not high. For example, when the electrode pair C that is not energized is adjacent to the downstream side of the electrode pair B, the sample solution is stopped near the boundary between the electrode pair B and the electrode pair C. As described above, by repeating the operation of suitably applying / stopping the voltage to the two pairs of electrodes, the sample liquid transfer control including transfer / stop can be realized, and the sample liquid can be reliably transferred to the destination.

  In the present invention, the sample solution can be reliably transferred to the destination even in the sample solution analysis chip in which three or more sets of N pairs (N = 3, 4, 5,...) Are arranged. In this case, first, after dropping the sample solution so as to at least partially overlap the first electrode pair and the second electrode pair, the voltage of the second electrode pair is more than the voltage of the second electrode pair. By applying a large voltage, the sample solution is transferred so as to spread over the activated second electrode pair, and the sample solution is caused to flow into the sample solution transfer path. Next, arbitrary three electrode pairs adjacent from the inlet side to the air outlet side in the sample liquid transfer path are selected. The arbitrary three electrode pairs may be selected from the two electrode pairs E and F arranged on both sides of the electrode pair D that is in contact with the tip of the sample solution. Of the three selected electrode pairs, a voltage higher than the voltage of the electrode pair D adjacent to the electrode pair E on the air inlet side is applied to the electrode pair E arranged on the most inlet side of the sample liquid transfer path. Call. As in the case of using two electrode pairs, it is preferable that the voltage of the electrode pair D be +2 to 3 V higher than the voltage of the electrode pair E. At this time, among the three electrode pairs, the electrode pair F arranged closest to the air opening is not energized (that is, voltage = 0) and is in an inactive state. Then, the sample liquid that is in contact with the electrode pair D and is stationary starts to move so as to expand the surface of the electrode pair D activated by energization, and the boundary between the electrode pair D and the inactive electrode pair F. It stops at the part. In this way, three electrode pairs are selected as appropriate, and the two electrode pairs are activated, and at the same time, the electrode pair arranged on the air port side of the two electrode pairs is adjacent to the air port side. By inactivating the pair of electrodes, the sample liquid can be reliably transferred to the destination while controlling the transfer / stop of the sample liquid. The operation of transferring / stopping the sample solution may be repeated up to the destination.

  In order to more reliably transfer the sample liquid to the destination, a sample liquid analysis chip having a sensing area may be used. The sensing area is an area where the position of the sample liquid to be transferred is sensed electrically or optically by the sensing means, and is set at an arbitrary position on the substrate. In addition, the sensing area may be configured by a member formed of a material having high light transmittance, like the detection area. The sensing means is not particularly limited, and may be the same as the detection means described above, or may be an electrode pair for transferring a sample liquid. In order to transfer the sample solution to the destination more reliably, it is preferable to install the sample solution in the vicinity of the upstream side of the destination to which the sample solution is transferred. Among the two electrode pairs that are adjacent to each other used for transferring the sample liquid and to which a voltage is applied, a preferred installation location is an electrode pair disposed on the air port side and further adjacent to the air port side than this electrode pair. In addition, a boundary portion with an inactive electrode pair to which no voltage is applied is included.

  When the position of the sample liquid is electrically sensed, (a) a step of preparing a sample liquid analysis chip having a sensing region for electrically sensing a change, (b) a first electrode pair and a second electrode (C) applying a voltage higher than the voltage of the second electrode pair to the first electrode pair partially overlapping the sample liquid; What is necessary is just to use the transfer control method which performs a process and the process of further detecting (d) an electrical change that the sample liquid contacted one electrode pair in the sensing area. Furthermore, in the case where three or more N pairs (N = 3, 4, 5,...) Of electrode pairs are arranged, any two pairs adjacent to each other along the flow of the sample liquid in the sample liquid transfer path. After detecting that the tip portion of the sample liquid in the sample liquid transfer path is in contact with the electrode pair B arranged on the air opening side of the electrode pair of the electrode pair, the sample liquid is applied to the electrode pair B. The step of applying a voltage higher than the voltage of the electrode pair B to the electrode pair A is repeatedly performed in a state of contact and stationary. The above-mentioned “electrical change” is sensed as a decrease in the resistance value between two pairs of electrodes to which a voltage is applied in order to control the transfer of the sample liquid. For example, when the sample solution contacts the end of the electrode pair B, the resistance value between the electrode pair A and the electrode pair B decreases. It is determined that the tip portion of the sample liquid has been transferred to the electrode pair B due to the decrease in the resistance value.

  When optically sensing the position of the sample liquid, (f) preparing a sample liquid analysis chip having a sensing region for optically sensing a change, (g) a first electrode pair and a second electrode Dropping the sample solution so as to at least partially overlap the electrode pair; (h) applying a voltage higher than the voltage of the second electrode pair to the first electrode pair partially overlapping the sample solution; A transfer control method for sequentially performing the steps and (i) the step of sensing as an optical change that the sample liquid has contacted one electrode pair in the sensing region may be used. Furthermore, in the case where three or more N pairs (N = 3, 4, 5,...) Of electrode pairs are arranged, any two pairs adjacent to each other along the flow of the sample liquid in the sample liquid transfer path. After detecting that the tip portion of the sample liquid in the sample liquid transfer path is in contact with the electrode pair B arranged on the air mouth side among the electrode pairs of The step of applying a voltage higher than the voltage of the electrode pair B to the electrode pair A in a state where the electrode pair A is in contact with and stationary may be repeated. At this time, in the sensing area, the position of the tip of the sample liquid is confirmed by sensing means including visual observation, and the sample liquid is appropriately transferred / stopped while confirming that the sample liquid has been transferred to the desired position. When such an operation is performed, the sample liquid can be sequentially transferred to the destination more reliably.

  In addition to the first sensing region for sensing the position of the sample solution, a second sensing region for sensing a change in the reaction reagent layer can be provided. The change of the reaction reagent layer means that the reaction reagent layer is dissolved or reacted in the sample solution. The second sensing region may be installed at the same location as the site where the reaction reagent layer is disposed or in the vicinity thereof. In order to more reliably observe the reaction behavior between the sample solution and the reagent that occurs in the reaction reagent layer, the second sensing region is located near the reaction reagent layer and downstream of the sample solution flow. It is preferable. The sensing means used in the second sensing area may be the same as the first sensing means described above, and includes visual observation.

[Analysis method using sample solution analysis chip]
Next, an analysis method using the sample liquid analysis chip according to the present invention will be described. In the following description, a mode in which the cholesterol concentration in plasma is measured using the sample solution analysis chip 10 shown in FIGS. 1 and 2 will be described. However, the sample liquid analysis chip of the present invention is not limited to the method for measuring cholesterol in blood, but can be used for other measurements as well.

The serum cholesterol value used as a diagnostic guide is the sum of the concentrations of cholesterol and cholesterol ester. As for the measurement of cholesterol, a measuring method for oxidizing cholesterol using an enzyme (cholesterol oxidase: ChOD) is generally used. However, since cholesterol ester cannot be a substrate for the oxidation reaction by ChOD, a method using cholesterol esterase (ChE) that converts cholesterol ester to cholesterol is known. That is, the serum cholesterol concentration is measured using the following reactions A and B using two kinds of enzymes.
Reaction A: Cholesterol ester → cholesterol + fatty acid (reaction with enzyme ChE)
Reaction B: Cholesterol + ferricyanide ion → cholestenone + ferrocyanide ion (reaction with enzyme ChOD)

  In this measurement, a reaction reagent layer mainly composed of ferricyanide ions is arranged in the first reaction reagent region 14a, and a reaction reagent layer mainly composed of ChE and ChOD is arranged in the second reaction reagent region 14b. At this time, for example, if a reaction reagent layer containing both ChE and ChOD is arranged in the first reaction reagent region arranged on the upstream side, reaction B by blood, ChE, and ChOD occurs. . This is because reaction B proceeds even if oxygen is present in the blood even in the absence of ferricyanide ions. When reaction B is started, oxygen is reduced and hydrogen peroxide is generated. Therefore, even if the ferricyanide ions in the second reaction reagent region 14b are subsequently reacted with blood, the ferricyanide ions are quantified. Cholesterol concentration cannot be measured accurately.

  First, blood, which is a sample solution, is dropped in the spotting portion in the sample supply unit A and near the inlet 25a of the sample solution transfer path 25. When blood is dropped, the first electrode pair composed of the electrodes 51 and 52 and the second electrode pair disposed on the air opening 25b side of the first electrode pair are in contact with each other. The sample solution is dropped into

  A voltage higher by about +2 to 3 V than the voltage of the second electrode pair is applied to the first electrode pair. Thereby, the blood spreads so as to cover the exposed portion of the second electrode pair and flows into the sample liquid transfer path 25. The blood that has flowed into the sample liquid transfer path 25 stops at the boundary between the second electrode pair and the third electrode pair. The blood contacts the first reaction reagent region 14a sandwiched between the second electrode pair. At this time, the first reaction reagent layer is dissolved in the blood, and the blood and potassium ferricyanide are mixed. However, since the ferricyanide ion cannot react with cholesterol or cholesterol ester by itself, the reagents are simply mixed in the first reaction reagent region 14a.

  In the first reaction reagent region 14a, after the blood and the reagent are sufficiently mixed, the voltage of the second electrode pair is +2 to 2 than the voltage of the third electrode pair constituted by the electrodes 55 and 56. Apply a voltage about 3V higher. Since the third electrode pair is an electrode pair adjacent to the air port 25b side of the second electrode pair, the blood stationary at the boundary between the first electrode pair and the second electrode pair is the third electrode pair. As a result, it is guided to the second reaction reagent region 14b sandwiched between the third electrode pair. In the second reaction reagent region 14b, blood dissolves in the second reaction reagent layer. Here, the components of the second reaction reagent layer 14b are mixed with the blood in which the components of the first reaction reagent region 14a have already dissolved, and a new reaction occurs.

  Since the second reaction reagent region 14b of this measurement is mainly composed of ChE and ChOD, cholesterol ester in the blood is changed to cholesterol by ChE. This blood is in a state where the changed cholesterol and the cholesterol originally contained in the blood coexist. Here, each cholesterol is oxidized to cholestenone by ChOD. In addition, potassium ferricyanide in the blood is reduced to potassium ferrocyanide.

  The second reaction reagent layer may not contain a surfactant so as not to increase the wettability of blood. In the absence of a surfactant, the rate of reaction of converting cholesterol ester to cholesterol by ChE is relatively slow. Therefore, although it depends on the concentration of ChE and its specific activity, the reaction time in the second reaction reagent layer requires at least about 10 minutes.

  After the reaction in the second reaction reagent region 14b is completed, a voltage higher by about +2 to 3V than the voltage of the fourth electrode pair constituted by the electrodes 57 and 58 is applied to the third electrode pair. The blood that has been stationary at the boundary between the second electrode pair and the third electrode pair spreads so as to cover the exposed part of the fourth electrode pair, and as a result, moves to the air port 25b side. Since the fourth electrode pair is formed to extend to the air port 25b, the blood reaches the air port 25b and stops.

  The blood is guided to the detection region 15 sandwiched between the fourth electrode pair on the substrate 11. The detection region 15 is not covered with a metal thin film for forming an electrode, and the substrate 11 having high light transmittance and electrical insulation is exposed. The cover 13 is made of a material having high light transmittance. Therefore, for example, by visually observing the color of the blood after reaction and comparing it with a sample of the color of blood before reaction prepared in advance, the degree of reaction between the blood and the reagent can be confirmed. . If the amount of potassium ferricyanide is measured by absorbance, potassium ferrocyanide can be quantified, and blood cholesterol concentration can be quantified. Further, the amount of ferricyanide ion decreased corresponds to the amount of cholesterol decreased by the oxidation reaction. Therefore, if the decrease amount of ferricyanide ions is quantified in the detection region 15, the cholesterol concentration can be calculated.

  If a reaction reagent layer containing a plurality of reagents is arranged in one place, the reagent deteriorates with the length of the storage period, so that the storage stability is lacking. On the other hand, if a plurality of reagents are arranged in different regions as described above, the storage stability of the reagents is remarkably improved. In addition, if the reagents are arranged in different regions, the order of the reactions can be controlled as appropriate, so that measurement with higher analysis accuracy is possible. Furthermore, if a plurality of reaction reagent regions are arranged with an arbitrary interval, the stored reagents will not be mixed with each other, so that the analysis accuracy is further improved.

  Next, a method for electrically detecting a change in the sample solution using the electrode will be described. When the change of the sample solution is electrically detected using the electrode, a sample solution analysis chip 100 as shown in FIG. 4 may be used. The sample liquid analysis chip 100 is the same as the sample liquid analysis chip shown in FIG. 1 except that a substrate to which a measurement electrode pair is added is used. Therefore, the same members as those described in the sample liquid analysis chip 10 are denoted by the same reference numerals, and description thereof is omitted. Further, illustration of the cover 13 having optical transparency is omitted. In the following description, an example of measuring the cholesterol concentration in plasma as a sample solution will be described.

  On the substrate 111 constituting the sample solution analysis chip 100, a third reaction reagent region 14c is formed in addition to the first reaction reagent region 14a and the second reaction reagent region 14b. The third reaction reagent region 14c is located closest to the air port 25b in the sample liquid transfer path 25, and is arranged so as to cover the surface of a measurement electrode pair 60 (see FIG. 5) described later.

  When the sample solution is plasma, it is preferable to dispose a reaction reagent layer mainly composed of potassium ferricyanide in the first reaction reagent region 14a and the second reaction reagent region 14b. Moreover, it is preferable to arrange a reaction reagent layer mainly composed of ChE and ChOD in the third reaction reagent region 14c. In the reaction reagent layer of the third reaction reagent region 14c located on the most downstream side of the sample liquid transfer path 25, a surfactant for improving the catalytic activity of ChE, such as Triton X-100, sodium cholate, octyl- β-d-thioglucoside and the like may be included. If the surfactant is mixed in the plasma, the wettability of the plasma with respect to the inner wall of the sample liquid transfer path 25 is improved, so that it becomes difficult to control the transfer of the plasma. By including a surfactant in the reaction reagent region 14c, it is possible to suppress as much as possible that the reagent necessary for the reaction flows out from the vicinity of the measurement electrode pair.

  As shown in FIG. 5, a measurement electrode pair 60 is formed on the substrate 111 constituting the sample liquid analysis chip 100 in addition to the first to fourth electrode pairs for controlling the transfer of plasma. Yes. The measurement electrode pair 60 includes a measurement electrode 61 and a counter electrode 62 which are a pair of electrodes, and is disposed on a part of the inner wall of the sample liquid transfer path 25, preferably on the bottom surface. By measuring the current value due to the oxidation reaction or reduction reaction of the substance in the sample liquid, or the substance generated by the chemical reaction between the substance in the sample liquid and the reaction reagent, by the measuring electrode 61 and the counter electrode 62, The concentration of certain substances can be detected. Grooves 77 and 78 are provided between the measuring electrode 61 and the adjacent electrode 58 and between the counter electrode 62 and the adjacent electrode 57, respectively.

  The measurement electrode 61 and the counter electrode 62 constituting the measurement electrode pair 60 are both planar electrodes made of a conductor or a semiconductor. The measurement electrode 61 is disposed at the center of the detection region 15. A counter electrode 62 is arranged in the vicinity of the measurement electrode 61 so as to surround the measurement electrode 61. The measuring station 61 and the counter electrode 62 may be formed as individually independent electrodes, or one conductor or semiconductor may be divided into two. Moreover, although the form which installed one electrode pair for a measurement was shown in this embodiment, the installation number of the electrode pair for a measurement is not specifically limited, What is necessary is just to install one or two or more.

  The width of the measurement electrode 61 is defined by the relationship between the electrode pair for sample liquid transfer and the width of the sample liquid transfer path 25. In order to fully exhibit the function as the measurement electrode 61 for detecting the change of the sample liquid, the width of the measurement electrode 61 is preferably 0.1 mm or more and 1.0 mm or less, more preferably 0.2 mm. It is 0.6 mm or less. In addition, the sample solution transfer electrode pair and the measurement electrode pair are preferably connected to an external terminal via a lead wire (lead wire). Thereby, it can connect easily with the electrochemical measuring apparatus etc. which were installed outside. As the conductor material, generally known conductors can be used. Among them, gold, platinum, silver, copper, palladium, and the like are preferably used.

  In accordance with the sample liquid transfer control method described above, the plasma dropped onto the sample liquid supply unit A is guided to the third reaction reagent region 14 c in the sample liquid transfer path 25. After the plasma is induced to the surface of the third reaction reagent region 14c, a pulse voltage is applied to the measurement electrode pair 60. At this time, a current value due to an oxidation reaction in which potassium ferrocyanide becomes potassium ferricyanide is measured. By quantifying the concentration of potassium ferrocyanide in plasma from the measured current value, the blood cholesterol concentration can be quantified.

[Method for Producing Sample Solution Analysis Chip of the Present Invention]
The sample liquid analysis chip of the present invention can be produced by any method as long as the effects of the invention are not impaired. Hereinafter, a preferred production method will be described.

  The sample liquid analysis chip of the present invention (A) detects an electrode pair for transferring a sample liquid on an insulating film-like substrate corresponding to the substrate and electrically or chemically changes the sample liquid as required. A step of forming a measurement electrode pair, (B) a step of forming a reaction reagent layer on the substrate, and (C) a pair of spacers arranged on both ends of the substrate, with the substrate and the cover interposed therebetween. And bonding them together to form a sample liquid transfer path with both ends released. Although the order in which each step is performed is not particularly limited, it is preferable that step (C) is finally performed from the viewpoint of workability.

  In the step (A), a plurality of electrode pairs for transferring the sample liquid and, if necessary, electrode pairs for measurement are formed on an electrically insulating substrate.

  As a method of forming the electrode pair, for example, a method of forming a disconnection portion between each electrode by forming a metal thin film on the surface of the substrate by sputtering or vapor deposition and then removing a part of the metal thin film. A method of removing a part of the metal thin film is a method of physically scraping with a ruter equipped with a file-shaped tool, or a method of irradiating a laser having a wavelength absorbed by the metal thin film to scatter the metal thin film in the irradiated portion. Can be used. Among them, the YAG laser is useful because it is not absorbed by PET but is absorbed by noble metals such as gold and palladium. If the electrodes are formed as described above, the disconnection portion in the insulating state formed by exposing the substrate can be formed in a linear shape, and therefore, by this line width, the shortest distance that can be taken as a gap between the electrodes is obtained. Is possible. Therefore, it is a very effective method when forming a plurality of electrode pairs.

  As another method of forming the electrode pair, there is a method of forming a metal thin film having a desired pattern on the substrate surface by sputtering or vapor deposition. Unlike the above-described method, this method forms a metal thin film on a substrate in a state where portions other than the portion corresponding to the planar shape of the electrodes constituting the electrode pair are covered with a mask plate. When this method is used, it is possible to form an electrode at an arbitrary position on the substrate and to accurately form a region not covered with the metal thin film. For example, if an electrode pair is formed on a transparent PET substrate and a metal thin film is not formed in the detection area, a sample liquid analysis chip that optically detects changes in the sample liquid in the detection area can be easily obtained. It is possible to get to.

  Furthermore, as another method for forming the electrode pair, there is a method in which a paste mixed with metal particles is applied to a substrate by screen printing. This method is a relatively convenient method that can be performed without a sputtering or vapor deposition apparatus. However, since the dimensional accuracy of the electrode shape is often slightly inferior to the two methods described above, it may not be suitable for forming a precise or fine electrode shape.

  When forming a measurement electrode pair which is a planar electrode, it can be formed by printing a paste containing a conductor or by using a known method such as sputtering or vapor deposition. The planar electrode may be formed by depositing gold, platinum, silver, copper, palladium, chromium, carbon, or the like in a thin film on a substrate surface such as polyethylene terephthalate. 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.

  In the step (B), a desired number of reaction reagent layers are formed on the substrate. The points to be noted regarding the location where the reaction reagent layer is disposed are as described above. The method for forming the reaction reagent layer is not particularly limited, and examples thereof include a method in which a reagent solution in which a reagent is dissolved in an arbitrary solvent is dropped on a substrate and then dried. When the reagent solution is solidified by drying, a layer containing a reagent, that is, a reaction reagent layer is formed. Here, the site where the reagent is dropped is preferably a site that becomes the inner wall of the sample liquid transfer path. More preferably, the position is the inner wall of the sample liquid transfer path on the substrate surface and is a non-electrode area sandwiched between the electrode pair for transferring the sample liquid or a position covering the detection area such as the measurement electrode pair. It is. For dropping the reagent solution, a pipette or a mechanical discharge device (dispenser) is suitable. For example, when a dispenser “SMPIII” manufactured by Musashi Engineering Co., Ltd. is used as the discharge device, it can be accurately dropped from about 50 nl to about 5 μl as a single dropping amount.

  Examples of the method of drying the reagent solution include a method of drying by blowing air (air-drying method). When performing the air-drying method, heated air can be used as long as the denaturation of the reagent does not become a problem. However, drying at room temperature is usually preferred in order to suppress denaturation of the reagent. Since the drying time varies greatly depending on the surrounding environment, the composition of the reagent solution, or the amount of the reagent solution dripped, it is appropriately determined according to each condition. Generally, a reaction reagent used in a sample liquid analysis chip often becomes a reaction reagent layer by drying for about 10 minutes to 1 hour.

  In addition, a freeze-drying method may be used for drying the reagent solution. The reaction reagent layer formed by the freeze-drying method has almost the same volume as the dropped solution, and has a void inside, so that it quickly dissolves into the sample solution. Furthermore, if a freeze-drying method for drying by sublimating the solvent is used, the amount of residual water in the reaction reagent layer after drying can be reduced as compared with the case of air drying in which the solvent is evaporated. Antibodies and the like can be stored without losing their properties.

The drying conditions in the freeze-drying method are not particularly limited as in the air-drying method, and may be appropriately selected according to the type of reagent solution. Hereinafter, an example of a procedure for drying about 1 μl of the dropped reagent solution by freeze-drying will be described.
(0) A reagent solution of about 1 μl is dropped at a predetermined position on the substrate surface.
(1) After dropping the reagent solution, the pre-frozen substrate is placed in an atmosphere of −40 ° C. and atmospheric pressure. At this time, the temperature inside the freeze dryer is set to −40 ° C., and the process waits until the substrate temperature actually becomes −40 ° C.
(2) The substrate cooled to −40 ° C. is placed in a vacuum environment and left at −40 ° C. for 15 minutes.
(3) Heat in a vacuum from −40 ° C. to −10 ° C. over 2 hours.
(4) Leave in vacuum at -10 ° C for 1 hour.
(5) Heat from −10 ° C. to 25 ° C. in vacuum over 2 hours.
(6) Leave in a vacuum at 25 ° C. for about 2 hours.

  In the step (C), the substrate and the cover manufactured in the steps (A) and (B) are bonded together via a spacer. When a pair of spacers are used, it is preferable that the spacers are arranged at the left and right ends on the substrate so as to face each other. The interval between the spacers is appropriately adjusted according to the desired width of the sample liquid transfer path. Moreover, when bonding a board | substrate and a cover, in order to raise adhesive strength, an adhesive agent and a hot press can also be used. Among these, an adhesive is useful from the viewpoint of ease of processing such as not requiring a large apparatus. As the adhesive, a known adhesive such as an acrylic resin as described above may be used.

[Example 1]
In Example 1, the sample liquid analysis chip 10 having the structure shown in FIG. 1 was used. Electrodes 51 to 58 made of a gold thin film were formed on the substrate 11. In the electrodes 51 to 58, the electrodes constituting the electrode pair are symmetrical with respect to the sample liquid transfer path, and the downstream end of each electrode along the sample liquid transfer path has a substantially triangular shape. And a pattern separated by grooves that form an acute angle with respect to the direction in which the sample liquid flows.

  This is a thin film made of gold by a sputtering device after a mask is made by punching a stainless steel plate through the shape of the desired electrode and then overlaying the mask on a transparent polycarbonate plate having a thickness of 0.2 mm. The electrodes 51 to 58 having different shapes were formed. The dimensions of the polycarbonate plate were 5.5 mm in width and 19 mm in length. In addition, the interval between the broken portions provided at the boundary between the electrode pairs was set to 0.1 mm. The electrodes 51 to 58 are arranged symmetrically with respect to the sample liquid transfer path, and are the distance between the electrodes constituting the electrode pair, and the width x2 of the non-electrode region that does not act as an electrode is 1.0 mm.

  The spacer 12 was disposed on the substrate 11 so that the width X of the sample liquid transfer path 25 was 1.4 mm and the length was 5.0 mm. At this time, an exposed portion having a desired size was formed in the sample liquid transfer path 25 by adjusting the arrangement of the spacers 12. Each electrode was exposed from the side wall of the sample liquid transfer path 25 toward the center side of the sample liquid transfer path 25. The exposed width x1 was 0.2 mm. The non-electrode region in the central portion of the sample liquid transfer path 25 is a region where a transparent polycarbonate substrate is exposed without an electrode being a gold thin film. Moreover, the terminal which can be connected to an external measuring device was formed in the edge part of each electrode 51-58.

  Most of the electrodes 51 and 52 constituting the first electrode pair were arranged inside the sample liquid supply unit A outside the sample liquid transfer path 25. On the other hand, the downstream ends of the electrodes 51 and 52 are projected toward the downstream side so as to reach the vicinity of the inflow port 25a. The shape of the ends of the electrodes 51 and 52 arranged in the vicinity of the inflow port 25a has a wedge shape as shown in FIG. The electrodes 53 and 54 constituting the second electrode pair are adjacent to each other via the electrodes 51 and 52 and the grooves 71 and 72, and are exposed to the outside of the sample liquid transfer path 25 and the sample liquid transfer path 25. Exposed parts. Further, of the electrodes 57 and 58 constituting the fourth electrode pair, a side of the third electrode pair that is on the inlet 25a side and is perpendicular to the flow of the sample liquid and parallel to the one side. The one side of the air port 25b was provided in close proximity. At this time, the distance between one side of the inlet 25a of the fourth electrode pair and the side of the third electrode pair on the air opening 25b side was made uniform. The end of the fourth electrode pair on the air port 25b side is arranged on the same line as the air port 25b.

  When assembling the sample liquid analysis chip, the shape of the exposed portion provided on the second and third electrode pairs is a parallelogram, and the shape of the fourth electrode pair is a trapezoid. The position of the spacer 12 was adjusted. The dimension of the exposed portion of the second and third electrode pairs is such that the long side forming a parallelogram, that is, the length of one side on the side of the sample liquid transfer path 25 is 1.3 mm; the short side forming a parallelogram That is, the length of one side protruding toward the center of the sample liquid transfer path 25 and forming an acute angle with respect to the sample liquid transfer path was about 0.66 mm. Further, the width x1 of the exposed portion was 0.2 mm. The width x1 of the exposed portion is the length of a perpendicular line from one side on the side wall of the sample liquid transfer path 25 to one side on the center side of the sample liquid transfer path 25.

  On the substrate 11, the first reaction reagent region 14 a and the second reaction reagent region 14 b were formed in the sample liquid transfer path 25 that does not act as an electrode. The 2nd reaction reagent area | region 14b was installed in the air port 25a side adjacent to the 1st reaction reagent area | region 14a. In the first reaction reagent region 14a of this example, a reaction reagent layer mainly composed of potassium ferricyanide shown below is disposed, and in the second reaction reagent region 14b, ChOD and ChE are mainly composed. A reaction reagent layer was placed. Details of the first reaction reagent region 14a and the second reaction reagent region 14b are shown below.

[First Reagent Area]
A mixed solution A containing 70 mmol / l potassium ferricyanide; 0.05 wt% carboxymethyl cellulose (CMC); taurine 1.33 wt%; maltitol 0.1 wt% was prepared. 0.1 μl of the prepared mixed solution A is dropped into a region on the substrate 11 where the sample liquid transfer path 25 is formed and the electrode sandwiched between the second electrode pairs is not formed to form a liquid film. did. This liquid film was dried and solidified in a laboratory at room temperature of 25 ° C. to form a reaction reagent layer in the first reaction reagent region 14a. This reaction reagent layer is a disc-shaped layer having a diameter of about 0.9 mm centered on an intermediate point between the regions sandwiched by the second electrode pair.

[Second reaction reagent area]
Nocardia-derived cholesterol oxidase (ChOD) 0.75 kilounits / ml (about 2.34 wt%); Pseudomonas-derived cholesterol esterase (ChE) 2 kilounits / ml (about 1.1 wt%); Taurine 1 wt%; A mixed solution B in which maltitol 0.25 wt%; CMC 0.2 wt% was dissolved in water was prepared. Next, in the region that becomes the sample liquid transfer path 25 on the substrate 11 and is sandwiched by the third electrode pair disposed on the air port 25b side adjacent to the first reaction reagent region 14a, 0.1 μl of the prepared mixed solution B was dropped to form a liquid film. This liquid film was dried in the same manner as the reaction reagent layer provided in the first reaction reagent region 14a to form a disc-shaped reaction reagent layer having a diameter of about 0.9 mm. This reaction reagent layer was also formed in a region where no electrode was formed.

  Two spacers 12 having a thickness of 50 μm, a length of 5.0 mm, and a width of 2.0 mm were prepared. An acrylic adhesive was applied to the surface of the PET substrate on which the substrate 11 and the cover 13 were bonded. After attaching the spacer 12 to a desired position on the substrate 11 so that the distance between the spacers 12 is 1.4 mm, a transparent polycarbonate cover 13 having a thickness of 100 μm is adhered to the spacer 12, thereby A liquid analysis chip 10 was formed.

[Measurement operation]
Plasma was used as the sample solution. 0.5 μl of plasma was dropped before the inlet 25a so as to be in contact with the inlet 25a. The dropped plasma is adjacent to the first electrode pair in the vicinity of the inflow port 25a, and the air port 25b side with respect to the first electrode pair, and a part of the plasma is transferred from the sample solution inflow port 25a. It was made to contact any electrode pair of the second electrode pair penetrating into the path 25. After visually confirming that plasma was appropriately dropped in the vicinity of the inlet 25a, a voltage of +2.5 V was applied to the first electrode pair rather than the voltage of the second electrode pair. Then, the plasma spreads quickly so as to cover the exposed portion provided in the second electrode pair, and eventually flows into the sample liquid transfer path 25, and then the second electrode pair and the third electrode pair Stationary at the border. When the color change of the first reaction reagent layer was visually observed, it was confirmed that the plasma contacted the first reaction reagent region and the reaction reagent layer was gradually dissolved in the plasma.

  The resistance value between the second electrode pair and the third electrode pair arranged on the downstream side adjacent to the second electrode pair is measured in advance by a measuring device to which each electrode is connected. As a result, the resistance value suddenly decreased. As a result, it was determined that the tip of the plasma reached the boundary between the second electrode pair and the third electrode pair, and that the tip of the plasma was in contact with the third electrode pair. The voltage application to the second electrode pair was stopped. The reason for the rapid contact of the plasma tip with a part of the third electrode pair is that the exposed part of the second electrode pair is the downstream end of the sample liquid transfer path 25 on the center side and the sample liquid transfer path 25. It seems that it originates in projecting toward the downstream side.

  The change in color of the reaction reagent layer in the first reaction reagent region was visually observed to determine the reaction condition between the reaction reagent layer and plasma. About 10 seconds after the reaction reagent layer and the plasma began to react in the first reaction reagent region, it was confirmed that the reaction reagent layer would be completely dissolved in the plasma due to a change in the color of the reaction reagent layer. Immediately after that, when a voltage + 2.5V higher than the voltage of the third electrode pair was applied to the second electrode pair, the plasma began to spread so as to cover the exposed portion of the third electrode pair, The reagent in the second reaction reagent region 14b started to dissolve while spreading in the center of the sample liquid transfer path 25 sandwiched between the three electrode pairs. In this case, the resistance value is measured in advance by setting a sensing region at the boundary between the third electrode pair and the fourth electrode pair arranged on the downstream side adjacent to the third electrode pair. As a result, the resistance value suddenly decreased. From this, it was determined that the plasma moved to the boundary between the third electrode pair and the fourth electrode pair, and the tip of the plasma was in contact with the fourth electrode pair. Application of voltage to the third electrode pair was stopped.

  In the plasma induced in the second reaction reagent region 14b, the reaction reagent layer provided in the first reaction reagent region 14a is dissolved. The tip of this plasma has moved to the region sandwiched between the third electrode pair, while the rear part of the plasma has not moved to the region sandwiched between the third electrode pair. Therefore, when the plasma was transferred to the downstream side, the rear part of the plasma moved to the air port 25b side as it was without dissolving the reaction reagent layer in the second reaction reagent region 14b. When the rear part of the plasma was observed visually, it was yellow derived from potassium ferricyanide contained in the reaction reagent layer of the first reaction reagent region 14a, so it is considered that potassium ferricyanide was contained at a high concentration.

  Although the vicinity of the second reaction reagent region 14b was visually observed, the phenomenon that the reaction reagent layer was dissolved in plasma could not be confirmed very clearly. Since the reaction reagent layer included in the second reaction reagent region 14b includes ChDH and ChE, ferricyanide ions included in the first reaction reagent region and cholesterol in plasma are included. It is presumed that the oxidation-reduction reaction of, and the reaction in which cholesterol ester in plasma by ChE is changed to cholesterol preceded by this oxidation-reduction reaction. In addition, in the sample solution analysis chip 10 of Example 1, since the surfactant for enhancing the activity of ChE is not added, it is determined that the reaction takes a long time, and the plasma is converted into the second reaction reagent region 14b. 10 minutes in a state of being moved to.

  After that, when a voltage +2.5 V higher than the voltage of the fourth electrode pair was applied to the third electrode pair, the plasma spread to cover the exposed portion of the fourth electrode pair, and the sample It moved further downstream in the liquid transfer path 25. Since the fourth electrode pair extends to the air port 25b, the plasma stopped here after reaching the air port 25b. At this time, in a region sandwiched between the fourth electrode pair on the surface of the substrate 11, there is a detection region 15 in which there is no gold thin film forming the electrode and the transparent and electrically insulating substrate 11 is exposed. Yes. In this example, since the cover 13 formed of a material having high light transmittance is used, the color of plasma was observed visually in the detection region 15. The cholesterol concentration could be calculated by comparing the observed plasma color with a color sample. A color sample is a color sample recorded in advance when a predetermined concentration of cholesterol is reacted. As described above, the prepared sample solution analysis chip 10 is formed of a material that can control the transfer of plasma suitably and has a high light transmittance despite the formation of a plurality of reaction reagent layers. By using the cover 13, good visibility was realized and the change in plasma reaction could be optically confirmed.

[Example 2]
In this example, plasma was used as the sample solution as in Example 1. Further, the measurement electrode pair 60 (measurement electrode 61, counter electrode 62) shown in FIG. 5 was used as a detection means for detecting the reaction between the sample solution and the reagent. By forming a palladium thin film on a 0.188 mm PET plate, which is the substrate 11, using a sputtering apparatus, and further providing a peeling portion on the palladium thin film with a line width of 0.1 mm with a YAG laser, FIG. The electrode 51 for measurement comprised from the electrode 51-58 of the shape of this, and the measurement electrode 61 and the counter electrode 62 was formed. The dimensions of this PET plate, the dimensions of the sample liquid transfer path, and the configuration of the substrate are the same as in the first embodiment. The arrangement of the electrodes on the substrate 11 and the electrode system for feeding the sample liquid are substantially the same as in the first embodiment, but the electrode pair that constitutes the electrode system is located on the most downstream side of the sample liquid transfer path. The shape of the fourth electrode pair composed of the electrodes 57 and 58 was changed from that in Example 1. The fourth electrode pair was disposed without extending the downstream end on the air port 25b side to the air port 25b. The counter electrode 62 was disposed adjacent to the electrodes 57 and 58 on the center side of the substrate 11. At this time, the width of the counter electrode 62 was made larger than the width of the sample liquid transfer path 25, and the air port 25 b was occupied by the counter electrode 62.

  The measurement electrode 61 is a central portion of the width of the sample liquid transfer path 25, and the end of the measurement electrode 61 on the air port 25b side is in contact with the air port 25b. The measurement electrode 61 was an ellipse electrode having a width of 0.6 mm and a length of 1.3 mm in the inflow direction of the sample liquid. In addition, a lead wire having a width of 0.15 mm is arranged on the measurement electrode 61 from the end on the air opening 25b side to the end of the substrate 11, and this lead wire is used as a terminal for connection with an external device. . The counter electrode 62 was disposed adjacent to the measurement electrode 61 so as to surround it. Similarly to the measurement electrode 61, the counter electrode 62 is provided with a lead wire from the end on the air opening 25b side to the end of the substrate 11 in order to form a terminal with an external device.

[First and second reaction reagent regions]
The positions where the first reaction reagent region 14a and the second reaction reagent region 14b are formed are exactly the same as in Example 1, but in this example, the same composition is applied to any of the reaction reagent regions 14a and 14b. That is, a reaction reagent layer mainly composed of potassium ferricyanide, which is an electron mediator, was disposed. A mixed solution C containing 70 mmol / l potassium ferricyanide; 0.05 wt% carboxymethylcellulose (CMC); taurine 1.33 wt%; maltitol 0.1 wt% was prepared. Then, 0.1 μl of the prepared mixed aqueous solution C was dropped in the vicinity of the inlet 25a of the sample liquid transfer path 25 to form a liquid film, and then dried to form a reaction reagent layer. The dropping of the mixed solution C and the drying conditions were the same as in Example 1.

[Third reaction reagent area]
The third reaction reagent region 14 c was arranged so as to cover the surface of the measurement electrode pair 60. In the present embodiment, the third reaction reagent region 14c acts as a detection region in addition to acting as a reaction region. The reaction reagent layer disposed in the third reaction reagent region 14c has a surface activity that promotes the activity of ChE for the purpose of shortening the reaction time between the reaction reagent layer and the sample solution in the third reaction reagent region 14c. The agent was added. When the plasma is dissolved in the reaction reagent layer containing the surfactant, the wettability of the plasma to the inner wall of the sample liquid transfer path 25 is improved. Therefore, even when no voltage is applied to the electrode pair, the plasma spontaneously flows to the air port 25b side. There is a risk of moving to. However, as in this embodiment, even when a reaction reagent layer containing a surfactant is provided in the third reaction reagent region 14c closest to the air port 25b, plasma is spontaneously generated in the adjacent reaction reagent region. Since the possibility of moving is low, the analysis accuracy is not impaired.

  The composition, shape, and formation method of the third reaction reagent region 14c are as follows. Cholesterol oxidase from Nocardia (ChOD) 0.75 kilounits / ml (about 2.34 wt%); Pseudomonas-derived cholesterol esterase (ChE) 2 kilounits / ml (about 1.1 wt%); and cholesterol esterase 0.5 wt% of polyoxyethylene-pt-octylphenyl ether (Triton X-100) which is a surfactant having an action to activate the reaction; 75 mmol / l of cholic acid sodium salt which is a surfactant; taurine 1 wt%; maltitol 0.25 wt%; CMC 0.2 wt% was mixed in water to prepare a mixed solution D. Next, 0.1 μl of the prepared mixed solution D was dropped on the central portion of the measurement electrode 61 and dried in the same manner as the reaction reagent layers arranged in the first and second reaction reagent regions. As a result, a disc-shaped reaction reagent layer having a diameter of 1.4 mm was formed around the measurement electrode 61.

[Measurement operation]
Plasma was supplied to the produced sample solution analysis chip 100 as a sample solution. As in Example 1, 0.5 μl of plasma was dropped before the inlet 25a so as to contact the inlet 25a. Further, the plasma is adjacent to the first electrode pair (electrodes 51 and 52) in the vicinity of the inflow port 25a, the air port 25b side with respect to this electrode pair, and a part of the plasma flows from the inflow port 25a to the sample liquid transfer path. The second electrode pair (electrodes 53 and 54) penetrating into 25 is in contact with any electrode pair.

  Immediately after that, when a voltage + 2.5V higher than the voltage of the second electrode pair is applied to the first electrode pair, the plasma spreads quickly so as to cover the exposed surface of the second electrode pair. As a result, after flowing into the sample liquid transfer path 25, the sample solution stopped at the boundary between the second electrode pair and the third electrode pair composed of the electrodes 55 and 56. In addition, a sensing area is set in advance between the second electrode pair and the third electrode pair adjacent to the air opening 25b side of the second electrode pair, and the resistance value in the sensing area is constantly measured. As a result, the resistance value suddenly decreased. At this time, when the inside of the sample liquid transfer path 25 was visually observed, the plasma tip contacted the upstream ends of the electrodes 55 and 56, and thus the current supply to the first and second electrode pairs was stopped. At this time, it was confirmed by the color change of the reaction reagent layer that the plasma also contacted the first reaction reagent region 14a sandwiched between the electrodes 53 and 54 and that the plasma was gradually dissolved in the reaction reagent layer. . The reason why a part of the plasma that is stationary at the boundary between the second electrode pair and the third electrode pair quickly contacts the third electrode pair is the same as in Example 1, as in the second electrode pair. It is surmised that the exposed portions of the electrodes 53 and 54 forming the shape are formed so that the downstream end portion on the central side of the sample liquid transfer path 25 protrudes toward the downstream side of the sample liquid transfer path 25.

  The change in color of the reaction reagent layer in the first reaction reagent region 14a was visually observed to determine the reaction condition between the reaction reagent layer and plasma. About 10 seconds after the reaction reagent layer and the plasma started to react, it was confirmed that the reaction reagent layer would be completely dissolved in the plasma, so that the third electrode pair was compared with the second electrode pair. A voltage higher by +2.5 V than the voltage was applied. Then, the plasma begins to spread so as to cover the exposed portion of the third electrode pair, reaches the second reaction reagent region 14b while spreading to the central portion of the sample liquid transfer path 25 sandwiched between the third electrode pair, The reaction reagent layer began to dissolve in plasma. In this case as well, when a sensing region was previously set at the boundary between the electrodes 55 and 56 and the electrodes 57 and 58 adjacent to the downstream side thereof, the resistance value was rapidly reduced. As a result, it was determined that the plasma reached the boundary between the third electrode pair and the fourth electrode pair, and the tip of the plasma was in contact with the fourth electrode pair. Application of voltage to the third electrode pair was stopped. The reaction reagent layer in the second reaction reagent region 14b had good solubility in plasma, similar to the reaction reagent layer in the first reaction reagent region 14a.

  Thereafter, a voltage +2.5 V higher than the voltage of the fourth electrode pair was applied to the third electrode pair. Then, the plasma spreads so as to cover the exposed portions of the electrodes 57 and 58 constituting the fourth electrode pair, and quickly contacts the third reaction reagent region 14c containing the enzyme and the surfactant before reaching the air port 25b. Reached. The reason why the plasma has moved to the air port 25b is considered to be that the shapes of the electrodes 57 and 58 constituting the fourth electrode pair are formed only up to the boundary with the third reaction reagent region 14c. It is up to the boundary between the third electrode pair and the fourth electrode pair that the plasma transfer can be controlled by improving the wettability by energization. However, since a part of the plasma comes into contact with the third reaction reagent region 14c and the reaction reagent layer is dissolved in the plasma, the surfactant contained in the third reaction reagent region 14c dissolves in the plasma, so that the plasma is dissolved. It is thought that it moved spontaneously to the air port 25b portion.

  Two minutes after the plasma reached the air port 25b, a voltage +400 mV higher than the voltage of the counter electrode 62 was applied to the measurement electrode 61 formed on the lower surface of the third reaction reagent region 14c. When a current value was measured 4 seconds after voltage was applied to the measurement electrode pair 60, a change in the current value that seems to depend on the cholesterol concentration could be measured. Further, as a result of performing the same measurement on the standard serum and a sample diluted with physiological saline, a current value depending on the cholesterol concentration could be observed as shown in FIG.

  Although the sample liquid analysis chip of the present invention has a simple structure, the sample liquid and the reagent can be rapidly and reliably analyzed in the chip. For this reason, the sample liquid analysis chip according to the present invention is useful as an analysis device for POCT such as a blood component measurement device.

(A) is schematic which shows an example of the form of the sample liquid analysis chip concerning this invention, (b) is sectional drawing which cut | disconnected the sample liquid analysis chip along the flow of the sample liquid. It is the schematic which looked at the sample liquid analysis chip | tip shown in FIG. 1 from the top. It is the schematic which expanded the sample solution transfer path vicinity among the board | substrates which comprise a sample solution analysis chip | tip. It is the schematic which shows an example of the form of the sample liquid analysis chip concerning this invention. It is the schematic which looked at the sample liquid analysis chip | tip shown in FIG. 4 from the top. It is a correlation diagram of the electric current value measured with the cholesterol sensor at the time of applying a sample liquid analysis chip | tip to a cholesterol sensor in Example 2, and the dilution rate of the sample liquid (plasma) used for the measurement.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Board | substrate 12 Spacer 13 Cover 14 Reaction reagent area | region 14a 1st reaction reagent area | region 14b 2nd reaction reagent area | region 14c 3rd reaction reagent area | region 15 Detection area | region 25 Sample liquid transfer path 25a Inflow port 25b Air port 51-58 Electrode 60 Measuring electrode pair 61 Measuring electrode 62 Counter electrode

Claims (11)

An electrically insulating substrate;
An electrode system including two or more pairs of a pair of electrodes formed on the substrate;
An electrically insulating cover bonded to the substrate via a spacer;
A sample flow path defined by the spacer between the substrate and the cover, the flow path having a sample liquid inlet and an air port provided on the opposite side of the inlet;
One or two or more reaction reagent regions formed in the sample solution transfer path on the substrate and arranged with a reaction reagent layer, and a detection region for detecting a physical or optical change of the sample solution;
A sample liquid supply unit formed on the substrate including the inflow port, wherein the sample liquid supplied to the supply unit is sucked from the inflow port into the sample liquid transfer path by capillary action A supply section;
A sample liquid analysis chip comprising:
The electrode system includes a first electrode pair disposed in the vicinity of the inlet, and a second electrode pair adjacent to the first electrode pair,
A part of the electrodes constituting the first electrode pair is formed outside the sample liquid transfer path from the inflow port,
The second electrode pair is formed closer to the air port side of the sample liquid transfer path than the first electrode pair,
Furthermore, when arranging three or more N pairs (N = 3, 4, 5,...) Of electrode pairs,
The N-th electrode pair is disposed adjacent to the (N-1) -th electrode pair on the air port side in the sample solution supply path with respect to the (N-1) -th electrode pair,
The electrodes constituting the electrode pair are symmetrical with respect to the sample liquid transfer path, and at least a part of the electrodes is disposed in a region where the sample liquid is supplied in the sample liquid supply unit, or in the sample liquid transfer path, A sample solution analysis chip that is not disposed in the reaction reagent region. The downstream end of the electrode constituting the electrode pair on the center side of the sample liquid transfer path protrudes toward the downstream side of the sample liquid transfer path,
2. The sample liquid analysis chip according to claim 1, wherein electrodes adjacent to each other along the sample liquid transfer path are separated through a groove extending in a direction that forms an acute angle with respect to a transfer direction of the sample liquid transfer path. The reaction reagent layer includes an oxidoreductase and an electron mediator,
2. The sample liquid analysis chip according to claim 1, wherein the detection area is disposed closer to the air port in the sample liquid transfer path than the reaction reagent area. The detection region is disposed on the surface of the substrate, and includes a measurement electrode pair composed of a measurement electrode and a counter electrode,
4. The sample liquid analysis chip according to claim 3, wherein the measurement electrode is arranged at a central portion of the detection region, and a width of the measurement electrode is narrower than a width of the sample solution transfer path.   The sample liquid analysis chip according to claim 4, wherein the measurement electrode is covered with the reaction reagent layer.   2. The sample liquid analysis chip according to claim 1, wherein at least a region that can serve as the detection region of the substrate or the cover is formed of a light-transmitting material.   2. The sample liquid analysis chip according to claim 1, wherein in the sample liquid transfer path, a sensing region for detecting the position of the tip of the sample liquid is disposed in the vicinity of a boundary between two adjacent electrode pairs. Preparing the sample liquid analysis chip according to claim 1;
Dropping the sample solution so as to at least partially overlap the first electrode pair and the second electrode pair;
Applying a voltage higher than the voltage of the second electrode pair to the first electrode pair in which the sample liquid partially overlaps,
Furthermore, when arranging three or more N pairs (N = 3, 4, 5,...) Of electrode pairs,
In the sample liquid transfer path, the sample liquid in the sample liquid transfer path is connected to the electrode pair disposed on the air port side among any two pairs of electrode pairs adjacent to each other along the flow of the sample liquid. With the tip part of the
A sample solution including a step of applying a voltage higher than the voltage of the electrode pair arranged on the air port side to the electrode pair on the inlet side of the sample solution transfer path among the two arbitrary electrode pairs. Transfer control method. Preparing the sample liquid analysis chip according to claim 7;
Dropping the sample solution so as to at least partially overlap the first electrode pair and the second electrode pair;
Applying a voltage higher than the voltage of the second electrode pair to the first electrode pair in which the sample liquid partially overlaps;
Sensing as an electrical change that the sample liquid is in contact with one electrode pair in the sensing region;
Have
Furthermore, when arranging three or more N pairs (N = 3, 4, 5,...) Of electrode pairs,
In the sample liquid transfer path, of the two pairs of electrodes adjacent to each other along the flow of the sample liquid, the sample in the sample liquid transfer path is connected to the electrode pair disposed on the air port side. After sensing by the sensing area that the tip of the liquid is in contact,
In a state where the sample solution is brought into contact with an electrode pair arranged on the air port side and is stationary,
A sample liquid transfer control method including a step of applying a voltage higher than the voltage of the electrode pair disposed on the air port side to the electrode pair disposed on the inlet side of the two sets of electrode pairs.   The sample liquid transfer control method according to claim 9, wherein the electrical change is sensed as a decrease in a resistance value between any two adjacent electrode pairs. Preparing the sample liquid analysis chip according to claim 7;
Dropping the sample solution so as to at least partially overlap the first electrode pair and the second electrode pair;
Applying a voltage higher than the voltage of the second electrode pair to the first electrode pair in which the sample liquid partially overlaps;
Sensing the contact of the sample liquid with one electrode pair in the sensing region as an optical change;
Have
Furthermore, when arranging three or more N pairs (N = 3, 4, 5,...) Of electrode pairs,
In the sample liquid transfer path, of the two pairs of electrodes adjacent to each other along the flow of the sample liquid, the sample in the sample liquid transfer path is connected to the electrode pair disposed on the air port side. After sensing by the sensing area that the tip of the liquid is in contact,
In a state where the sample solution is brought into contact with an electrode pair arranged on the air port side and is stationary,
A sample liquid transfer control method including a step of applying a voltage higher than the voltage of the electrode pair disposed on the air port side to the electrode pair disposed on the inlet side of the two sets of electrode pairs.

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