JP2011107099A - Electrochemical analysis chip and electrochemical analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrochemical analysis chip which can perform electrochemical analysis even with respect to a substance to be measured, which can not be directly subjected to electrochemical analysis, such as protein or the like by a simple method and enabling accurate quantitative analysis, and an electrochemical analyzing method. <P>SOLUTION: The electrochemical analysis chip, which is used in the electrochemical analysis related to the substance to be measured which cannot be subjected to direct electrochemical detection, includes a laminated structure including a sample absorbing layer, a reagent layer and a sample measuring electrode in this order from above. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an analysis chip used for electrochemical analysis. More specifically, the present invention relates to an analysis chip used for electrochemical analysis of a substance to be measured that cannot be directly detected electrochemically.

  In recent years, the importance of detecting, detecting or quantifying biological substances such as DNA, enzymes, antigens, antibodies, proteins, viruses and cells, and chemical substances has increased in the fields of medicine, health, food, and drug discovery. Various biochips and microchemical chips (hereinafter collectively referred to as analysis chips) that can easily measure them have been proposed. The analysis chip is designed so that a series of experiments and analysis operations performed in the laboratory can be easily performed within a chip of several centimeters square and several millimeters to 1 cm thick. Therefore, it has many advantages such as low cost, high reaction rate, high throughput testing, and the ability to obtain test results immediately at the site where the sample is collected. Such an analysis chip is suitably used for biochemical tests such as blood tests.

  Currently, various methods are used for simple measurement of various substances derived from living bodies. In the medical field such as influenza test, pregnancy test, blood glucose level measurement, etc., it is simple and inexpensive, the demand for general public is high, and it has gained a big market. It is expected to form an increasingly large market in the future.

  Patent Document 1 discloses an electrochemical test device for measuring the presence or concentration of an analyte in an aqueous liquid sample. As a specimen in a specific aqueous liquid sample, glucose in blood is described. By using such an electrochemical test device, it is possible to perform quantification by a simple operation as long as it is a glucose concentration in blood or the like.

  On the other hand, some corticoids such as cortisol are known as hormones whose body concentration changes due to stress. Currently, in the actual measurement of such proteins and the like, qualitative inspection is performed visually for the presence or absence of coloration or color reaction of reagents held on paper-based test chips, or the intensity of coloration or color development is optically detected. The mainstream method is to perform quantitative measurement by measuring the target. However, with a physiologically active substance such as cortisol, which is present in a very small amount in a biological sample, when a paper-based test chip is used, it is difficult to perform accurate protein quantification because of insufficient sensitivity.

Moreover, many proteins, because it is rare for generating a simple low molecular weight molecules in the reaction system, be linked to the consumption of redox highly of H ₂ O ₂ generating and O ₂ as glucose As a result, it is difficult to perform quantitative analysis from the transfer of electrons using electrochemical measurements. In that case, quantitative analysis cannot be performed unless a complicated method such as an enzyme-linked immunosorbent assay (ELISA) is used. For this reason, analysis chips for accurately quantitatively analyzing proteins and the like by a simple, rapid and low-cost method such as an electrochemical analysis chip for blood glucose have hardly been put to practical use.

Special table 2001-518620 gazette

  In view of the present situation as described above, the present invention can perform an electrochemical analysis by a simple method even on a substance to be measured such as a protein that cannot be directly subjected to an electrochemical analysis, and is accurate. An object is to provide an analysis chip and an analysis method capable of quantitative analysis.

  The present invention relates to a substance to be measured that cannot be directly detected electrochemically, having a laminated structure including a sample absorption layer, a reagent layer, and a sample measurement electrode in this order from above. It is an analysis chip used for electrochemical analysis.

  The chemical reaction necessary for the electrochemical analysis preferably includes an antigen-antibody reaction with the substance to be measured.

It is preferable that the antibody is disposed near the surface of at least a part of the electrode.
Furthermore, an antibody immobilization layer in which an antibody is immobilized in a state in contact with the electrode between the electrode and the reagent layer is provided, and at least a portion of the antibody immobilization layer corresponding to the surface of the electrode has an antibody. It is preferable that they are arranged.

  The reagent layer includes a complex of the substance to be measured and an electrochemically detectable label substance (hereinafter, a complex of the substance to be measured and an electrochemically detectable label substance is measured substance-labeled substance). (Abbreviated as a composite).

  The labeling substance is preferably glucose oxidase or glucose dehydrogenase.

The substance to be measured is preferably a corticoid or a corticoid derivative.
Furthermore, it is preferable to have a mechanism for removing liquid at the bottom of the laminated structure.

  Furthermore, it is preferable to include a blank measurement electrode and / or a standard (standard solution: solution having a known concentration of the substance to be measured) measurement electrode.

Furthermore, it has a flow path for the sample (measuring solution: solution of unknown concentration of the substance to be measured) and a developing cleaning solution,
The sample measuring electrode and the blank measuring electrode may be disposed within the flow path of the sample and the developing cleaning liquid, and the standard measuring electrode may be disposed outside the flow path of the sample and the developing cleaning liquid. preferable.

  It is preferable that the antibody is disposed near the surface of the sample measurement electrode and the standard measurement electrode, and the antibody is not disposed near the surface of the blank measurement electrode.

  It is preferable that a cover member having an opening for dropping the sample and the developing cleaning liquid is provided above the laminated structure.

Moreover, it is preferable to provide the said several laminated structure.
In addition, the present invention moves the sample containing the substance to be measured and the developing cleaning solution from the upper side to the lower side of the laminated structure using the analysis chip having the laminated structure including the sample absorption layer, the reagent layer, and the electrode in this order from above. The present invention also relates to a method for electrochemical analysis of a substance to be measured that cannot be directly detected electrochemically, wherein a pretreatment reaction necessary for the electrochemical analysis is performed.

  According to the analysis chip of the present invention, it is possible to easily perform electrochemical analysis of proteins and the like, and to speed up the analysis. In addition, if it is possible to perform accurate quantification not only quickly and qualitatively at a price equivalent to the current paper base, it can be used for screening to determine immediately whether the disease is serious, As a point-of-care testing (POCT) technology, it can greatly contribute to the medical field.

It is a schematic cross section which shows an example of the laminated structure of the analysis chip of this invention. It is a schematic cross section which shows another example of the laminated structure of the analysis chip of this invention. It is a figure which shows an example of the analysis chip of this invention. (A) is a schematic cross-sectional view, and (b) is a schematic top view. It is a figure which shows another example (multi analysis type) of the analysis chip | tip of this invention. (A) is a schematic cross-sectional view, and (b) is a schematic top view. 3 is a graph showing the results of electrochemical analysis of Example 1. 3 is a graph showing the results of electrochemical analysis of Example 1. 6 is a graph showing the results of electrochemical analysis of Example 2. 6 is a graph showing the results of electrochemical analysis of Example 3.

  For example, glucose present in blood or the like generates an oxidation-reduction current by a reaction catalyzed by the enzyme glucose oxidase or the like, and can be directly detected electrochemically by measuring this current. On the other hand, many of various proteins existing in living bodies are difficult to electrochemically quantify and qualify by causing a reaction that directly generates a redox current. For this reason, when measuring proteins and the like in living bodies using electrochemical measurement, the substance to be measured and the substance to be measured-labeled substance complex undergo a competitive reaction, and the labeling substance is used for electrochemical reaction. The detection method is used automatically. The present invention is an analysis chip for simply performing an electrochemical analysis of a substance to be measured that cannot be directly detected electrochemically through other indirect processing reactions.

  Specific examples of analytes that cannot be directly detected electrochemically include bioactive substances such as proteins such as enzymes, physiologically active substances such as nucleic acids and hormones, neurotransmitters, and low molecular weight compounds. It is done. Preferred are low molecular weight compounds, and more preferred are corticoids and derivatives thereof. Examples of the corticoid include cortisol, corticosterone, cortisone and the like, preferably cortisol.

  The analysis chip is a handy size measuring member used for analyzing a sample containing a substance to be measured (for example, detecting or quantifying the substance to be measured in the sample) by adopting an arbitrary measurement principle. The analysis chip of the present invention has a laminated structure including at least a sample absorption layer, a reagent layer, and a sample measurement electrode in this order from above.

  An example of the laminated structure of the analysis chip of the present invention will be described with reference to FIG. FIG. 1 shows a structure in which a sample measurement electrode 21 is disposed in a recess on a substrate 1, a reagent layer 3 is further stacked thereon, and a sample absorption layer 41 is further stacked thereon.

  The sample absorption layer 41 is a member that absorbs a sample containing a substance to be measured, and the sample and the developing cleaning liquid are developed on the reagent layer 3 disposed below by absorbing a developing cleaning liquid described later. . The constituent material of the sample absorption layer 41 is a material that can absorb the sample and is not particularly limited as long as it does not denature the substance to be measured. Examples thereof include cellulose, glass fiber, and silica fiber. Preferred are cellulose and glass fiber.

  The shape of the sample absorption layer 41 is not particularly limited as long as the liquid can flow in the vertical direction, and examples thereof include a porous body, a pad made of a fiber material, a membrane, and the like. Although the thickness of the sample absorption layer 41 is not specifically limited, 100 micrometers-3 mm are preferable, More preferably, they are 100 micrometers-1 mm.

  The reagent layer is a layer in which a necessary reagent is held on a carrier capable of flowing a liquid vertically. The material of the carrier is not particularly limited as long as it does not denature the substance to be measured, and examples thereof include glass fiber, cellulose fiber, and silica fiber. Glass fiber and cellulose fiber are preferable. Various known methods can be used as the method for holding the reagent on the carrier, and examples include a method in which a solution containing the reagent is infiltrated into the carrier and then dried. In addition, it is preferable that a reagent is hold | maintained by moderate intensity | strength so that it may be flowed to the electrode vicinity with a developing washing | cleaning liquid.

  The shape of the carrier is not particularly limited as long as the liquid can flow in the vertical direction, and examples thereof include a porous body, a pad made of a fiber material, a membrane, and the like. Although the thickness of a reagent layer is not specifically limited, 100 micrometers-3 mm are preferable, More preferably, they are 100 micrometers-1 mm.

  The reagent held in the reagent layer is a reagent used for a pretreatment reaction necessary for performing an electrochemical analysis of a substance to be measured. Although the pretreatment reaction necessary for the electrochemical analysis of the substance to be measured will be described later, for example, a competitive reaction may be mentioned. Examples of the reagent used when the pretreatment reaction is a competitive reaction include a substance to be measured-labeled substance complex in an antigen-antibody reaction. When this complex and the substance to be measured are made to compete with each other, when introduced onto the electrode, an antigen-antibody reaction with an antibody arranged near the electrode surface occurs, and the label of the complex bound to the antibody is detected by electrochemical analysis. As a result, the substance to be measured can be quantitatively determined.

  Note that only one type of reagent may be held in one reagent layer, or two or more types of reagents may be held.

  The sample absorption layer 41 and the reagent layer 3 do not necessarily need to be separate members, and necessary reagents are held in the lower part of the integral carrier. It may be an integral structure that also serves as

  The sample measurement electrode usually includes a working electrode (working electrode), a counter electrode (counter electrode), and a reference electrode (reference electrode). When performing an electrochemical analysis, after flowing a sample containing a substance to be measured and a developing cleaning solution, in a certain state, a current change with respect to a potential change of the working electrode with respect to the reference electrode (flowing between the working electrode and the counter electrode) Current change) is detected. Analyze the substance to be measured (quantitative, qualitative) by comparing the electric quantity (coulomb quantity) or peak current value or differential value of the current change obtained by integrating the current change with the calibration curve. Can do.

  As the material for the working electrode, various known working electrode materials used for electrochemical analysis of a labeling substance can be used, and examples thereof include platinum, carbon, gold, silver, palladium, ruthenium, and rhodium. In particular, when the reagent is a substance to be measured-labeled complex and glucose oxidase is used as the labeling substance, it is preferable to use a working electrode containing platinum as a material for the detection reaction (oxidation reaction by glucose oxidase). . The working electrode in the present invention includes an electrode in which an antibody is immobilized on the surface of the working electrode as described later, and an electrode containing an electron mediator.

  As the material of the counter electrode, various known counter electrode materials used for electrochemical analysis of the labeling substance can be used, and examples thereof include platinum, carbon, gold, silver, palladium, ruthenium, and rhodium. Platinum is preferable.

  As the material of the reference electrode, various known reference electrode materials used for electrochemical analysis of the labeling substance can be used, and examples thereof include platinum, carbon, gold, silver, palladium, ruthenium, and rhodium. Platinum is preferable.

  The material of the substrate 1 is not particularly limited. For example, polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene ( PE), polyethylene naphthalate (PEN), polyarylate resin (PAR), acrylonitrile butadiene styrene resin (ABS), vinyl chloride resin (PVC), polymethylpentene resin (PMP), polybutadiene resin (PBD), biodegradation Organic materials such as conductive polymer (BP), cycloolefin polymer (COP), and polydimethylsiloxane (PDMS); inorganic materials such as silicon, silicone, glass, and quartz can be used.

  The substrate surface is preferably provided with a recess (wiring pattern) for arranging a sample measurement electrode (including a working electrode, a counter electrode, and a reference electrode), a blank measurement electrode described later, a standard measurement electrode, and the like. The method for forming the recess is not particularly limited, and examples thereof include an injection molding method using a mold having a transfer structure, an imprint method, and the like. In the case of forming a substrate using an inorganic material, an etching method or the like can be used.

  In the analysis chip of the present invention, the laminated structure may include other layers as necessary. For example, as shown in FIG. 2, an antibody fixing layer 5 described later is disposed above the sample measurement electrode 21. You may provide in contact (under the reagent layer 3).

  The size of the analysis chip of the present invention is not particularly limited, and can be, for example, about several cm in length and width and about several mm to 1 cm in thickness.

  The analysis chip of the present invention has a structure (vertical flow structure) for flowing a sample and a developing cleaning liquid in the vertical direction. When performing an electrochemical analysis, the sample containing the substance to be measured and the developing cleaning liquid are stacked. By moving from the upper side to the lower side of the structure, a pretreatment reaction necessary for performing an electrochemical analysis of the substance to be measured is performed.

  When the substance to be measured is contained in a liquid component derived from a living body such as saliva, sweat or blood, the liquid component derived from the living body can be used as a sample as it is. Among these liquid components derived from living organisms, saliva and sweat are preferably used as samples in that they can be collected without invasion. In the case where contaminants in the liquid component become a problem, a liquid dispersed or dissolved in the same medium as a developing cleaning liquid described later may be used as a sample after performing processing such as extraction or centrifugation. . From the viewpoint of simplicity of analysis, it is preferable to use a liquid component derived from a living body as a sample.

  The developing cleaning liquid is a liquid having both a role as a developing liquid and a role as a cleaning liquid. The role as a developing solution is to develop the substance to be measured in the sample absorption layer on the reagent layer, and further on the antibody immobilization layer (if necessary) or on the electrode. The pretreatment reaction necessary for the electrochemical analysis of the measured substance is advanced. The role as a cleaning liquid is, for example, a role of washing away excess substance to be measured and substance to be measured-label complex not adsorbed on the antibody and removing them from the electrode and the antibody fixing layer.

  The developing washing liquid is appropriately selected according to the type of the substance to be measured, the kind of the substance to be measured-labeled complex, and the antibody. For example, as a developing washing liquid used when the substance to be measured is corticoid and the substance to be measured-labeled complex is a corticoid-glucose oxidase complex, phosphate buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, Tris buffer, acetate buffer and the like. In particular, when the corticoid is cortisol, it is preferable to use a phosphate buffer as a developing cleaning solution.

  Usually, the sample is first introduced into the sample absorption layer and then developed into the sample absorption layer, and then the developed cleaning solution is introduced into the sample absorption layer, whereby the substance to be measured is developed into the reagent layer, and further the electrode. It is developed on an antibody or antibody immobilization layer arranged in the vicinity of the surface.

  In the present invention, since the analyte to be analyzed cannot be directly subjected to electrochemical analysis, a sample pretreatment reaction using a separate chemical reaction is required in advance to perform electrochemical analysis. It is. Examples of the pretreatment reaction necessary for performing this electrochemical analysis include an antigen-antibody reaction.

  In the laminated structure of the analysis chip, when the pretreatment reaction necessary for performing the electrochemical analysis includes an antigen-antibody reaction using the substance to be measured as an antigen, the electrode can detect the current and perform the electrochemical analysis. In order to do so, the antibody is preferably disposed in the vicinity of the surface of the electrode. Specifically, it is preferable to fix the antibody directly on the surface of at least a part of the electrode.

  Further, as shown in FIG. 2, in addition to the above laminated structure, an antibody fixing layer 5 to which an antibody is fixed is further provided between the electrode 2 and the reagent layer 3, and at least a part of the surface of the electrode is provided. The antibody may be arranged at the corresponding position above, and in this case, more antibodies can be arranged than when the antibody is directly fixed to the electrode. In order to increase the current generated in the electrode and improve the measurement sensitivity, it is preferable that the antibody fixing layer 5 is disposed in contact with the electrode 2.

  The antibody fixed on the surface of the electrode or the antibody fixing layer needs to be fixed so as not to flow at least by the sample and the developing washing liquid. Various known immobilization methods can be used as the antibody immobilization method.

  The antibody immobilization layer is a layer in which an antibody used for an antigen-antibody reaction using a substance to be measured as an antigen is immobilized on a carrier through which a liquid can flow in the vertical direction. The material of the carrier is not particularly limited as long as it does not modify the substance to be measured, and examples thereof include nitrocellulose, polyvinylidene fluoride (PVDF), and silica. Nitrocellulose is preferred.

  The shape of the carrier is not particularly limited as long as it allows the liquid to flow in the vertical direction through the carrier. In the case where a nitrocellulose membrane is used as the carrier, the shape of the carrier is preferably circular. This is because when a cutting process is performed with a cutter or the like, a crack or the like is likely to occur at the cut portion, and therefore, it is easier to process with a punch. Although the thickness of an antibody fixed layer is not specifically limited, 0.1 mm-0.5 mm are preferable, More preferably, it is 0.2 mm-0.3 mm.

  The analysis chip of the present invention further includes a blank measurement electrode and / or a standard measurement electrode for measuring a standard in the laminated structure of the analysis chip including the sample measurement electrode or separately from the laminated structure. It is preferable to provide an analysis chip that can simultaneously measure a sample and a blank and / or a standard. At this time, the sample measurement electrode and the blank measurement electrode are preferably arranged in the flow path of the sample and the developing cleaning liquid, and the standard measurement electrode is arranged outside the flow path of the sample and the developing cleaning liquid. preferable. The number of standard measurement electrodes can be increased as necessary, and may be omitted if not necessary.

  For example, as shown in FIG. 3, a sample measurement electrode 21 and a blank measurement electrode 22 are arranged on a substrate 1, and a first reagent layer 31 is laminated thereon, and a sample absorption layer 41 is further laminated thereon. A laminated structure in which the standard measurement electrode 23 is arranged on the substrate 1 at a position apart from the laminated structure, the second reagent layer 32 is laminated thereon, and the standard absorbing layer 42 is laminated thereon. The partition wall 6 can be provided between the two stacked structures. In FIG. 3, a cover member 7 having an opening 71 for dropping the sample and the developing cleaning liquid and an opening 72 for dropping the standard (standard solution) and the developing cleaning liquid is provided above the laminated structure. It has been.

  Each of the blank measurement electrode and the standard measurement electrode usually includes a working electrode, a counter electrode, and a reference electrode, similarly to the sample measurement electrode. As the material for the working electrode, the counter electrode, and the reference electrode, the same materials as those used for the above-described sample measurement electrode can be used. However, the antibody is not usually fixed or the antibody fixing layer is not provided on the surface of the working electrode of the blank measurement electrode.

  The substance to be measured in the sample measurement electrode and the standard measurement electrode by immobilizing an antibody or providing an antibody immobilization layer on the surface of the working electrode of the sample measurement electrode and the standard measurement electrode Can be measured. On the other hand, on the surface of the working electrode of the blank measuring electrode, the antibody is not fixed or the antibody fixing layer is not provided, so that the current change of the working electrode of the blank (when no substance to be measured is present) is changed. Can be detected.

  The standard measurement electrode 23 is disposed on the substrate 1 separately from the sample measurement electrode 21 and the blank measurement electrode 22, and the antibody is directly immobilized thereon or an antibody immobilization layer is disposed thereon. The reagent layer 32 soaked with the reagent is disposed thereon, and the standard absorption layer 42 is disposed thereon, whereby the standard used for calibration can be measured. Therefore, in the standard absorption layer 42 on the standard measurement electrode 23, it is necessary to impregnate the standard, not the sample, and the sample measurement electrode 21 and the blank measurement electrode 22 are placed between the sample measurement electrode 21 and the blank measurement electrode 22 so that the adjacent sample is not mixed. It is preferable to provide the partition wall 6.

  The analysis chip of the present invention prepares a necessary number of electrodes and arranges a combination of a reagent layer, a sample absorption layer, an antibody immobilization layer, and the like in a single analysis chip from multiple samples. A type (multi-analysis type) analysis chip capable of simultaneously measuring a plurality of substances to be measured may be used.

  An example of a multi-analysis type analysis chip will be described with reference to FIG. 4, the sample measurement electrode 21, the blank measurement electrode 22, the standard measurement electrode 23, the first reagent layer 31, the second reagent layer 32, the sample absorption layer 41, and the standard absorption layer shown in FIG. 42 and the part including the partition wall 6 are shown as measurement parts 81, 82, 83, 84. Each of the measuring units 81, 82, 83, and 84 includes a reagent, an antibody, and the like for measuring different types of substances to be measured, and can measure a plurality of types of substances to be measured. In addition, a partition 9 is provided between the measurement units 81, 82, 83, and 84 so that a sample containing a plurality of types of substances to be measured and a developing cleaning liquid do not mix with each other between the measurement units. ing.

  In FIG. 4, in such a multi-analysis type analysis chip, openings provided in the cover member 7 for dropping the sample and the developing cleaning liquid (opening 71 for dropping the sample, opening for standard dropping) A plurality of units 72) are provided depending on the type of electrode of each measuring unit.

  The analysis chip of the present invention preferably includes a mechanism for draining surplus samples, developing cleaning liquid, and the like. Examples of the mechanism for draining such excess liquid include an absorption pad and a waste liquid tank. When using an absorbent pad, cellulose, a glass filter, etc. can be used as a material of the absorbent pad.

  The present invention also relates to an electrochemical analysis method using the above-described analysis chip. The electrochemical analysis method of the present invention is an electrochemical analysis method of a substance to be measured that cannot be directly detected electrochemically, and has a laminated structure including a sample absorption layer, a reagent layer, and an electrode in this order from above. A pretreatment reaction necessary for the electrochemical analysis is performed by moving the sample containing the substance to be measured and the developing cleaning liquid from the upper side to the lower side of the laminated structure using the analysis chip.

  In addition, as a process after performing the said pretreatment reaction, the process of detecting the label | marker of the to-be-measured substance-label complex couple | bonded with the antibody by the method similar to a well-known electrochemical analysis is mentioned, for example. Specifically, for example, when the labeling substance is glucose oxidase, by supplying a certain amount of glucose to the vicinity of the electrode surface through the sample absorption layer, an oxidation reaction with glucose oxidase is performed, and the current change at that time is changed. By measuring, the adsorption ratio of the substance to be measured and the substance to be measured-labeled complex to the antibody can be analyzed, and the substance to be measured can be quantified from the result.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

Example 1
<Antibody immobilization layer>
1) A nitrocellulose membrane (Trans-Blot (registered trademark) Transfer Medium Pure Nitrocellulose Menbrane (thickness 0.45 μm), 162-0145, Bio-Rad Laboratories Inc.) is cut into a 3 × 6 mm rectangle, and 3 with distilled water. Washed twice. In addition, the washing | cleaning method put the washing | cleaning liquid (distilled water) into the Eppendorf tube for the number of washing | cleaning times 1mL at a time, and wash | cleaned by putting the film | membrane in it. Thereafter, the same washing is performed.

  2) A nitrocellulose membrane was immersed in 400 μL of a cortisol antibody solution (1 μg / mL, 10 μg / mL, and 0 μg / mL as a control) and incubated at room temperature for 90 minutes.

  3) After washing 3 times with a phosphate buffer solution (hereinafter abbreviated as PBS-T) containing 0.05% of 10% Tween 20 (10% Tween 20 Solution, 161-0781, Bio-Rad Laboratories Inc.) as a surfactant. The sample was immersed in 1 mL of a blocking solution (1 μg / mL BSA solution) and incubated at 4 ° C. for 90 minutes. Furthermore, it wash | cleaned 3 times by PBS-T and obtained the nitrocellulose membrane (antibody fixed layer) by which the cortisol antibody was fixed.

<Pretreatment reaction>
The antibody immobilization layer is immersed in 500 μL of a solution of glucose oxidase and cortisol complex (hereinafter abbreviated as GOD-CORT complex) (100 μL of 0.106 mg / mL GOD-CORT complex solution + 400 μL of PBS-T). The antibody reaction was allowed to proceed. After 30 minutes, the plate was washed 5 times with PBS-T to remove unreacted substances.

<Measurement method>
1) Place the treated nitrocellulose membrane on the platinum electrode, add 35 μL of distilled water, and use an electrochemical analyzer (Electrochemical Analyzer Model 832A, ALS832A, ALS / chi) to 0.6V (Pt ) Was applied for measurement.

  2) After the current value was stabilized, 5 μL of a 100 mg / dL glucose solution was added, and current measurement was started immediately after that. The measurement results are shown in FIG. FIG. 5 is a graph showing the relationship between the detection current value and the measurement time in the cortisol antibody solution (1 μg / mL, 10 μg / mL, and 0 μg / mL as a control) having the above-mentioned concentrations.

  As shown in FIG. 5, since the detection current value increases as the concentration of the cortisol antibody increases, the cortisol antibody is immobilized on the nitrocellulose membrane, and the antigen-antibody reaction between the cortisol antibody and the GOD-CORT complex is specific. Was confirmed to be happening.

  In Example 1 above, when the concentration of the cortisol antibody solution is 10 μg / mL, a GOD solution (0.1 mg / mL) solution or distillation is used instead of the GOD-CORT complex solution (0.106 mg / mL). Using water (control), the current value was measured in the same manner. The results are shown in FIG. As shown in FIG. 6, it can be seen that when the GOD solution (0.1 mg / mL) is used, the current value is hardly detected, but in the case of the GOD-CORTO complex, the current value is detected. Therefore, it was confirmed that the antigen-antibody reaction was specifically performed between the GOD-CORT complex and the cortisol antibody.

(Example 2)
<Antibody immobilization layer>
A nitrocellulose membrane (antibody immobilization layer) on which the cortisol antibody was immobilized was prepared in the same manner as in Example 1 except that a 10 mg / mL cortisol antibody solution was used.

<Pretreatment reaction>
100 μL of Cortisol standard solution (1 ng / mL, 10 ng / mL) was added to 500 μL of GOD-CORTO complex solution (100 μL of 0.106 mg / mL GOD-CORT complex solution + 400 μL of phosphate buffer), and the cortisol obtained above. The competitive reaction was allowed to proceed by immersing the nitrocellulose membrane on which the antibody was immobilized. After 30 minutes, the plate was washed 5 times with PBS-T to remove unreacted substances. As a control, a solution obtained by immersing a nitrocellulose membrane on which a cortisol antibody was immobilized only in 500 μL of the GOD-CORTO complex solution was also prepared.

<Measurement method>
About the nitrocellulose membrane which performed the said process, it carried out similarly to Example 1, and measured the electric current value. The measurement results are shown in FIG. FIG. 7 is a graph showing the relationship between the detected current value and time when the cortisol standard solution of each concentration of 1 ng / mL and 10 ng / mL was added and when the cortisol standard solution was not added as a control.

  In FIG. 7, when the concentration of the cortisol standard solution increases, the detected current value decreases, indicating that the concentration of cortisol standard solution and the current value have an inversely proportional relationship. Therefore, it was confirmed that a competitive reaction between cortisol and GOD-CORT complex was performed on the nitrocellulose membrane. Therefore, it can be said that cortisol can be quantified in the laminated structure including the antibody immobilization layer and the electrode in this order from above, and it is considered that the same quantification is possible in the analysis chip having the laminated structure of the present invention. .

(Example 3)
<Antibody immobilization layer>
A nitrocellulose membrane (HF180UBXSS manufactured by Millipore) was cut with a punch into a circular shape having a diameter of 6 mm. 10 μL of 0.1 mg / mL of cortisol antibody was dropped on the cut antibody fixing layer (nitrocellulose membrane) and dried at room temperature. Next, the antibody fixing layer is immersed in 20 mL of a blocking solution for 60 minutes. The blocking solution used here is a solution containing PBS-T and bovine serum albumin (BSA) in a weight ratio of PBS-T: BSA = 100: 2. Next, the operation of removing the nitrocellulose membrane and immersing it in PBS-T 20 mL for 5 minutes for washing was repeated 3 times, and then dried at room temperature.

<Reagent layer>
A grafibre conjugate pad (GFCP103000 manufactured by Millipore) was cut with a punch into a circular shape having a diameter of 6 mm. 5 μL of a solution containing a complex of glucose oxidase and cortisol (GOD-CORT complex) at an excessive concentration was dropped onto the cut-out glass fiber conjugate pad and dried at room temperature to obtain a reagent layer.

<Sample absorption layer>
A cellulose fiber sample pad (CFSP203000 manufactured by Millipore) was cut into a circular shape having a diameter of 6 mm with a punch to obtain a sample absorption layer.

<Assembly of analysis chip>
As shown in FIG. 2, the antibody fixing layer, the reagent layer, and the sample absorption layer prepared as described above are placed on a sample measurement electrode 2 (including a working electrode, a counter electrode, and a reference electrode) made of a platinum (Pt) material. The antibody fixing layer 5, the reagent layer 2, and the sample absorption layer 3 were laminated in this order from the bottom. In addition, the absorption pad for absorbing a surplus sample, a developing washing | cleaning liquid, and glucose aqueous solution was installed separately by the side surface vicinity of the laminated structure formed in this way.

<Standard analysis>
5 μL of standard having a cortisol concentration of 1 ng / mL or 10 ng / mL in which cortisol is dissolved in pure water is absorbed by the sample absorption layer. Thereafter, 500 μL of PBS-T is slowly dropped from above the sample absorption layer as a developing cleaning solution. After dropping the entire amount of the developing cleaning solution and stabilizing the response current, 10 μL of an aqueous glucose solution (100 mg / dL) is added dropwise. During this period, the surplus sample, the developing cleaning solution, and the glucose aqueous solution are absorbed by the absorption pad.

  Electrochemical analysis was started immediately after the start of dropping of the aqueous glucose solution, and a graph showing the relationship between the detected current and the measurement time was created. The resulting graph is shown in FIG.

<Experimental result>
In the graph shown in FIG. 8, the solid line is a measurement result of a standard solution having a cortisol concentration of 1 μg / mL, and the broken line is a result of an electrochemical measurement in which a standard solution having a cortisol concentration of 10 μg / mL is measured. . As shown in FIG. 8, the result that the current value of the output was smaller as the cortisol concentration was larger was obtained. Accordingly, it can be said that cortisol can be quantified using a competitive method in a laminated structure including a sample absorption layer, a reagent layer, an antibody immobilization layer and an electrode in this order from above.

  It should be understood that the embodiments and examples disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

  1 substrate, 21 sample measurement electrode, 22 blank measurement electrode, 23 standard measurement electrode, 3 reagent layer, 31 first reagent layer, 32 second reagent layer, 41 sample absorption layer, 42 standard absorption layer, 5 Antibody immobilization layer, 6,9 partition, 7 cover member, 71, 72 opening, 81, 82, 83, 84 measurement part.

Claims (13)

  Electrochemical analysis of a substance to be measured that cannot be directly detected electrochemically, having a laminated structure including a sample absorption layer, a reagent layer, and a sample measurement electrode in this order from above Analysis chip used for the test.   The analysis chip according to claim 1, wherein an antibody is arranged near the surface of at least a part of the electrode.   And an antibody immobilization layer in which an antibody is immobilized in contact with the electrode between the electrode and the reagent layer, and the antibody is present in at least a portion of the antibody immobilization layer corresponding to the surface of the electrode. The analysis chip according to claim 1, which is arranged.   The analysis chip according to claim 1, wherein the reagent layer contains a complex of the substance to be measured and an electrochemically detectable labeling substance.   The analysis chip according to claim 4, wherein the labeling substance is glucose oxidase or glucose dehydrogenase.   The analysis chip according to claim 5, wherein the substance to be measured is a corticoid or a corticoid derivative.   Furthermore, the analysis chip in any one of Claims 1-6 which has a mechanism which removes a liquid in the lower part of the said laminated structure.   The analysis chip according to claim 1, further comprising a blank measurement electrode and / or a standard measurement electrode. Furthermore, it has a flow path for the sample and the developing cleaning liquid,
The sample measurement electrode and the blank measurement electrode are disposed in the flow path of the sample and the developing cleaning liquid, and the standard measurement electrode is disposed outside the flow path of the sample and the developing cleaning liquid. Analysis chip as described in.   The analysis chip according to claim 8 or 9, wherein the antibody is disposed near the surface of the sample measurement electrode and the standard measurement electrode, and the antibody is not disposed near the surface of the blank measurement electrode.   The analysis chip according to any one of claims 1 to 10, further comprising a cover member having an opening for dropping the sample and the developing cleaning liquid above the laminated structure.   The analysis chip according to claim 1, comprising a plurality of the laminated structures.   Using the analysis chip having a laminated structure including the sample absorption layer, the reagent layer, and the electrode in this order from above, the sample containing the substance to be measured and the developing cleaning liquid are moved from above to below the laminated structure. A method for electrochemical analysis of a substance to be measured that cannot be directly detected electrochemically, wherein a pretreatment reaction necessary for analysis is performed.

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