JPH03164195A - Analysis of liquid specimen and apparatus therefor - Google Patents

Abstract

PURPOSE:To quickly analyze plural components in serum such as hormones at the same time by contacting a liquid specimen with a reagent-containing layer and measuring the variation of the volume of generated gas by an optical detection means utilizing a surface plasmon phenomenon through an adjacent gas-permeable layer. CONSTITUTION:A liquid specimen (e.g. serum) is introduced through a specimen injection port of an analytical instrument 1 and made to contact with a reagent- containing layer 3 holding an enzyme (e. g. glucose oxidase). If an analytical object (e. g. glucose) is present in the liquid specimen, gas is generated and/or consumed according to the progress of enzymatic reaction. The variation of the volume of the gas is transmitted through a gas-permeable layer 4 adjacent to the reagent-containing layer 3 and retained on the active surface 5 of an optical structure. The liquid specimen can be analyzed by irradiating a light beam 9 from a light source 8 to the active surface 5 and measuring the variation of the reflecting light beam 10 with a detector 11 utilizing a surface plasmon phenomenon.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a liquid sample analysis method and analysis device that utilizes the surface plasmon phenomenon. [Prior Art] There are various methods for testing liquid samples such as serum to determine the presence and/or concentration of various substances, such as hormones, vitamins, lipids, enzymes, nitrogen-containing substances, sugars, and antigenic substances. It is becoming increasingly important from the perspective of early detection of diseases. Among these methods, methods that utilize gases produced or consumed by enzymatic reactions include methods that electrochemically measure gas changes using oxygen electrodes, and methods that colorimetrically analyze pigments produced from gas and color-forming substances. etc. are commonly used. Furthermore, there is also the so-called enzyme immunomethod, which basically takes advantage of the specificity of the immune reaction and uses an enzyme reaction as a detection means. However, conventional enzymatic reaction methods have complicated processes and low detection accuracy. Another problem with enzyme immunoassays, which have high detection accuracy, is that they take too much time. On the other hand, an analysis method using surface plasmon resonance is also known. For example, W○ (PCT International Publication) 8 8/0
6 7 2 5 and 8 8/0 6 7 2 6 each describe the use of surface plasmon resonance to quantify gases contained in a liquid sample (for example, blood). However, the gases that are measured using the techniques described in these specifications are oxygen gas, carbon dioxide gas, or anesthetic gas originally contained in the blood sample. That is, there is no mention of measuring gases converted from sugars in blood through enzyme reactions for the purpose of testing. Furthermore, a technique for capturing and analyzing the occurrence of antigen-antibody reactions of trace components contained in blood in the form of surface plasmon resonance is disclosed in Japanese Patent Application Laid-open Nos. 61-29045 and 63-2716.
It is stated in Publication No. 2. However, this technology is a complex process and requires a long time. Furthermore, these patent publications do not describe capturing gas changes. [Problems to be Solved by the Invention] The present inventors have discovered that by measuring gas changes caused by enzyme reactions, for example, by utilizing the surface plasmon phenomenon, it is possible to quickly, accurately, and easily determine the components in a liquid sample. We discovered that analysis can be performed using a simple process. Furthermore, the present inventors found that performing the above analysis on a rotatable disk not only enables more efficient analysis but also has excellent effects such as the ability to analyze multiple parts at the same time. I also found out what you can get. The present invention is based on these findings. [Means for Solving the Problems] According to the present invention, the change in gas amount caused by bringing a reagent into contact with a liquid sample can be controlled by utilizing the surface plasmon phenomenon through a gas permeable layer. This can be achieved by measuring optically. That is, according to a specific aspect of the present invention, a liquid sample is brought into contact with an enzyme- or substrate-containing layer, and the change in gas amount due to the enzyme reaction is measured by adjusting the gas permeability of the layer adjacent to the enzyme- or substrate-containing layer. The method is characterized in that the measurement is carried out through the layer using an optical detection means that utilizes the surface plasmon phenomenon.
A method for analyzing liquid samples [hereinafter sometimes referred to as the enzyme method]. The invention also provides: contacting a mixture of an enzyme-labeled first immunoreactive substance and a liquid sample with a layer containing a second immunoreactive substance;
The first immunoreactive substance that did not bind to the second immunoreactive substance and the liquid sample are removed, the substrate for the labeled enzyme is added, and the change in gas amount due to the influence of the enzyme reaction is measured to the second immunoreactive substance. A method for analyzing a liquid sample [hereinafter sometimes referred to as enzyme immunoassay], characterized in that measurement is carried out by an optical detection means using a surface plasmon phenomenon through a gas-permeable layer adjacent to a substance-containing layer. It also relates to The present invention also provides the following features: a liquid reagent injection means; a reagent-containing layer disposed to be in contact with the liquid sample injected by the injection means; a gas permeable layer; a surface plasmon active region; a surface plasmon active region for directing light to the surface plasmon active region. The present invention also relates to a liquid sample analysis device characterized in that it includes a means for irradiating; and an optical detection means using a surface plasmon phenomenon at the active surface. Furthermore, according to a preferred embodiment of the present invention, there is provided a method for carrying out the above-mentioned enzyme method, enzyme immunoassay, etc. on a rotatable disk, or an analytical device for bringing a reagent into contact with a liquid sample on a rotatable disk. Provided. The "optical detection means using surface plasmon phenomenon" used in the present invention includes, for example, r Sur
face plasmon resonance fo
r gas detection and biosens
sing J (Nylander et al., Sanso
rs and Actuators, 4, 229-304.
, 1983), there is a method of measuring the change in permittivity caused by a change in the gas composition as a change in the plasmon resonance angle, and
nced RamanVibrational Stu
dies at SoliaGas Interface
es” (by Pockrand, Sprunger V
There is a method of determining changes in gas amount using Raman absorption, which is described in ``Erlag''. Also, what is Raman absorption using surface plasmon phenomenon (
(Although there is not always a consensus on this), specifically, light is irradiated onto the surface of a substrate coated with silver, gold, copper, etc., and Raman absorption of substances existing near the surface is performed. means a method of measuring or a similar method. The present invention will be explained in detail below. The analysis method of the present invention uses a reagent that generates or consumes gas when it comes into contact with a liquid sample, and the amount of gas at this time is optically detected using surface plasmon phenomenon at a position through a gas permeable layer. It is measured by means.

Specifically, these include, but are not limited to, the enzymatic method or enzyme immunoassay described below, and reagents that cause gas changes due to reactions upon contact with specific components in liquid samples such as blood. This includes the case of For example, hemoglobin in blood becomes oxyhemoglobin by reacting with an oxygen reagent, and at this time oxygen is consumed.

Furthermore, since hydrogen peroxide in water generates oxygen when it comes into contact with manganese dioxide, it can be measured by the present invention. Below, typical aspects of the present invention will be explained in detail. In the enzyme method of the present invention, the specificity of the enzyme or substrate contained in the enzyme- or substrate-containing layer is utilized to detect a certain component to be tested contained in the liquid sample. Measure the presence and/or concentration (content or activity i) of Enzyme- or substrate-containing layers can be prepared by loading (trapping or especially immobilizing) enzymes or substrates on porous materials. Porous materials include textiles made of fibrous materials such as synthetic fibers (e.g. polyamide), natural fibers (e.g. cotton or wool), inorganic fibers (e.g. glass fibers);
It can be in the form of a filter made of knitted or especially non-woven fabrics or porous inorganic or organic materials. Enzymes can be supported (immobilized or captured) on this porous material by a known method, such as impregnation or coating with an enzyme or substrate suspension. Enzymes that can be used in the present invention are those that produce or consume gases (eg, hydrogen peroxide, oxygen, carbon dioxide, or ammonia) through enzymatic reactions. Specific examples of these enzymes include various oxidases, such as glucose oxidase (COD), galactose oxidase, cholesterol oxidase, choline oxidase, uricase (urate oxidase), lactate oxidase, oxalate oxidase, pyruvate oxidase, and ascorbate oxidase. , amino acid oxidase, alcohol oxidase, monoamine oxidase; decarboxylase, such as oxalate decarboxylase, amino acid decarboxylase; deaminase, such as adenosine deaminase, AMP deaminase; or,
Examples include creatinase, ammonia-lyase, and urease. The enzymes described above can be used to detect glucose, sucrose, maltose, galactose, cholesterol or cholesterol esters, phosphatidylcholine, triglycerides (triglycerides) in liquid samples.
, urea, uric acid, lactic acid, oxalic acid, pyruvic acid, ascorbic acid, amino acids, alcohol, creatinine, adenosine, adenosine monophosphate, adenosine triphosphate,
The presence and/or concentration of monoamines etc. can be analyzed. Here, if the test substance is sucrose, for example, sucrose is converted to α-1D-glucose using invertase, which is further converted to β-1D-glucose using mutarotase, and then glucose oxidase is applied. As it consumes oxygen and simultaneously produces hydrogen peroxide,
All you have to do is measure the changes in these gases. In this way, test substances that are preferably changed in advance into a form that can be used in enzymatic reactions that involve gas changes include maltose, galactose, cholesterol or cholesterol esters, phosphatidylcholine, and neutral fats (triglycerides). ．． These test substances may be used as samples in the method of the present invention after being changed in advance using an appropriate enzyme into a form that can be used in enzymatic reactions that involve gas changes. It is also possible to carry an enzyme or a group of enzymes to be transformed into a usable form together with the enzyme-containing layer, and to transform the enzyme in the enzyme-containing layer. The liquid sample used in the method of the present invention may be any liquid that may contain the specific substance to be tested, and in particular may be a biological liquid sample, such as a body fluid, such as blood (whole blood, serum) or urine. In addition, the substances to be tested are substances that serve as substrates for enzymes. For example, sugars (e.g. glucose)
, organic acids, lipids, amino acids, proteins or bebutides,
Furthermore, enzymes such as glutamate monooxaloacetate transaminase (GOT) or glutamate monopyruvate transaminase (GPT).

In the method of the present invention, changes in gas amount are measured through a gas permeable layer. The gas-permeable layer is prepared from a material that acts as a barrier to substances other than gaseous substances and does not allow them to pass through, but allows only gaseous substances to pass through.
Such materials include, for example, cellulose acetate butyrate (10 to 60% by weight of butyryl groups and 5 to 30% of acetyl groups).
Examples include materials such as cellulose probionic acid valerate (containing 20 to 50% by weight of valeryl groups), acetylated cellulose acetate (containing 19% or more of acetyl groups), polytetrafluoroethylene, or silicone rubber. ．． In the method of the present invention, changes in the amount of gas are measured using the surface plasmon phenomenon, and in this case, an active surface that can maintain the surface plasmon phenomenon generated by light irradiation can be used. An optical structure having such an active surface has at least a portion made of a metal such as silver, gold, or copper, and has an optimal uneven shape (pitch, depth, cross-sectional shape, etc.) depending on the wavelength or angle of the incident light. have. For example, optical structures having surfaces with only irregularities, in particular diffraction grating surfaces with a suitable pitch, preferably metallized. Furthermore, prisms with metal coating surfaces, metal fine particles (especially about 0.01 to 10μ
Microparticle) dispersions of m can also be used as active surface-supported optical structures. This metal-coated surface usually transmits light. The active surface carried on the optical structure is typically isolated from the outside air by an air seal. The outside air sealing means is, for example, a gas detection chamber consisting of a space surrounded by an outer wall, and a support layer (for example, made of plastic or glass) provided adjacent to the optical structure. Alternatively, if the optical structure consists of an active surface portion (metallic coating) and its support portion (prism), for example a prism with a surface carrying a metal coating, the support portion (prism) may be It can also be used as a means of sealing off the outside air. When the optical structure is installed within the gas detection chamber, the optical structure itself may be in contact with the gas-permeable layer or separate from it. The support layer is a light-transmitting layer that stably maintains the gas state on the non-plasmon active surface. Therefore, it is not necessary if the permeated gas from the gas-permeable layer can remain in a certain constant state at the surface. In the method of the present invention, first, a liquid sample is brought into contact with an enzyme-containing layer. If the liquid sample contains a substance that produces or consumes gas by the enzyme, an enzymatic reaction occurs and the amount of gas changes. For example, when glucose oxidase is immobilized on the enzyme-containing layer and serum is used as the liquid sample, if glucose is present in the serum, an enzyme reaction [blank below] glucose gluconic acid +28202 occurs, oxygen is consumed, and Hydrogen peroxide is produced. ``I. When urea and urease are combined, 82CO3 (H20 + CO2) + 2NH3 ammonia and carbon dioxide gases are generated.Changes in the amount of gases such as this occur quantitatively (i.e., liquid (corresponding to the amount of a specific substance in the sample) is retained in the surface plasmon active part of the active surface of the optical structure. To measure the amount of gas with the optical structure, for example, irradiated to generate surface plasmon phenomenon,
All you have to do is observe the reflected light or transmitted light (including modulated light). In the enzyme immunoassay of the present invention, a specific component contained in a liquid sample is captured by utilizing the specificity of the antigen-antibody reaction, and then an enzyme is added as a label. to determine the presence and/or concentration of that particular fraction. The enzyme immunoassay of the present invention can be carried out using a competitive assay or a sandwich assay. In competitive testing methods, the same antigen as the antigen to be measured is labeled with an appropriate enzyme and used as the first immunoreactive substance. On the other hand, an antibody specific to this antigen is used as a second immunoreactive substance and is supported on a porous material. Furthermore, in the sandwich test method, an antibody against the antigen to be measured is labeled with an appropriate enzyme and used as the first immunoreactive substance. On the other hand, an antibody that binds to another site of this antigen is used as a second immunoreactive substance and is supported on a porous material. Antibodies used in competitive or sandwich tests are:
Monoclonal antibodies prepared by cell fusion technology are preferred, but polyclonal antibodies prepared from antiserum may also be used. As the porous material, the materials described in the above enzyme method can be used, and the second antibody can be supported on the porous material by a known method, for example, by impregnating or coating with an antibody suspension. Can be done. Enzymes that can be used in the present invention are enzymes that generate or consume gases (e.g., hydrogen peroxide, oxygen, carbon dioxide, or ammonia) through enzymatic reactions, and are specifically applicable to the enzymatic method described above. As mentioned above.
These enzymes are used as labels for the first immunoreactive substance (antigen or antibody). In the enzyme immunoassay of the present invention, the same gas permeable layers and optical structures used in the enzymatic methods described above can be used. In the enzyme immunoassay of the present invention, a mixture of a liquid sample that may contain the antigen to be tested and a first immunoreactive substance labeled with an enzyme is brought into contact with a second immunoreactive substance-containing layer. After sufficient time has elapsed to terminate the antigen-antibody reaction, the liquid sample and the enzyme-labeled first immunoreactive substance that are not bound to the second immunoreactive substance are removed with a suitable detergent.Continued When a liquid containing a substrate for a labeled enzyme is added, an enzymatic reaction will occur if the liquid sample contains the antigen to be tested, and the gas amount will change (e.g. due to consumption of oxygen and hydrogen peroxide). This change in the amount of gas is retained on the active surface of the optical ellipse quantitatively (i.e., corresponding to the amount of a specific substance in the liquid sample) through the gas-permeable layer. Next, by irradiating light and making use of the surface plasmon phenomenon, the measurement can be carried out in the same manner as the enzyme method described above. When the enzyme method described above is carried out using the analyzer of the present invention, a liquid sample is introduced from the liquid sample injection means. After the completion of the washing, an appropriate washing solution can be introduced. When carrying out the enzyme immunoassay described above, in addition to introducing the liquid sample from the liquid sample injection means,
Introduce a wash solution to remove immunoreactive substances, unbound substances, and excess liquid sample, as well as a substrate-containing solution. Also, after the analysis of a certain liquid sample is completed, an appropriate cleaning solution can be introduced. Therefore, in this specification, "liquid sample" refers to a liquid sample to be analyzed, as well as a liquid containing an enzyme-labeled first immunoreactive substance,
It contains substrate-containing liquid, etc. The analysis device of the present invention has a biologically reactive substance-containing layer disposed so as to be in contact with the liquid sample injected by the injection means. Here, "biologically reactive substances" refer to enzymes or substrates (when carrying out the above-mentioned enzymatic methods) and second immunoreactive substances, namely antibodies or antigens (when carrying out the above-mentioned enzyme immunological methods). Therefore, the biologically reactive substance-containing layer is prepared by containing the enzyme/substrate or antibody/antigen in the porous material. In addition, it is equipped with a surface plasmon phenomenon measuring means consisting of a suitable light source and a detector.Next, in accordance with FIGS. The analysis principle when implementing the analysis method of the present invention using the analyzer of the present invention will be explained. To implement the enzyme method of the present invention, first, the sample injection port (not shown) of the analyzer 1 is ) is introduced into the liquid sample 2 and brought into contact with the enzyme-supporting layer 3. If a test substance that reacts with the enzyme in the enzyme-supporting layer 3 is present in the liquid sample 2, the enzyme reaction progresses and gas is produced and/or consumed. This change in the amount of gas is carried through the gas-permeable layer 4 to the active surface 5 on the optical structure (not shown). It is protected from the effects of the outside air by a detection chamber 7 (FIG. 2).A light source 8 irradiates the active surface 5 with a light beam 9, and the absorption change or polarization change of the reflected light beam 10 is measured by a detector 11. As shown in Figure 2,
When the support layer 12 is provided on the outside of the gas detection chamber 7, the support layer can be set to any shape or size, such as a disk shape, for example.

Next, in order to carry out the enzyme immunoassay of the present invention, first, a liquid sample and an enzyme-labeled antigen (in the case of a competition method) or an antibody (in the case of a sandwich method) are added to the sample injection port (not shown) of the analyzer 1. Introduce mixture 2 with case 2 and bring it into contact with antibody-supporting layer 3. After the time required for antigen-antibody reaction has elapsed, wash solution is introduced from the sample injection port to remove the mixture 2 that binds to the antibody on antibody-supporting layer 3. Remove the liquid sample and the enzyme-labeled antigen or antibody that are not present.Next, when a liquid containing a substrate for the labeled enzyme is poured from the sample injection port, if the enzyme-labeled antigen or antibody is bound to the antibody on the antibody-supporting layer 3. The enzymatic reaction progresses to generate and/or consume gas.Changes in the amount of gas are measured in the same manner as described above.According to a preferred embodiment of the present invention, the enzyme described above The method and the enzyme immunoassay are carried out on a rotatable disk, or a part of said analytical device is constructed on a rotatable disk.That is, according to a preferred embodiment of the invention, at least Introducing a liquid sample from the center of the disk in an analysis test section extending from the center toward the circumference, and causing the liquid sample to flow in the direction of the circumference by centrifugal force generated by rotation of the disk. The change in the amount of gas caused by the enzyme reaction is detected through a gas-permeable layer (preferably adjacent to the enzyme/substrate-containing layer) using an optical method utilizing surface plasmon phenomenon. According to a preferred embodiment of the present invention, at least one portion of the rotatable disk is arranged from the center to the circumference. In the analytical test section extending towards the
A mixture of an immunoreactive substance and a liquid sample is introduced from the center of the disk, and the mixture is caused to flow circumferentially by centrifugal force generated by rotation of the disk, thereby containing a second immunoreactive substance. the first immunoreactive substance and the liquid sample that did not bind to the second immunoreactive substance are removed, and the substrate for the labeled enzyme is added from the center of the disk and the rotation of the disk is continued. The substrate is brought into contact with the second immunoreactive substance-containing layer by centrifugal force, and the change in gas amount due to the influence of the enzyme reaction is controlled by a gas-permeable layer (preferably adjacent to the second immunoreactive substance-containing layer). A method for analyzing a liquid sample is provided, which is characterized in that measurement is performed using an optical detection means that utilizes the surface plasmon phenomenon. In the above preferred embodiment, the biologically reactive substance-containing layer and the gas permeable layer are usually provided adjacent to each other, but an intermediate layer may be provided between them for pH adjustment, etc. Good too. Furthermore, according to a preferred embodiment of the invention, there is provided a rotatable analytical disk comprising: an analytical test section extending from the center towards the circumference on at least a portion of the disk; a liquid sample provided in the center of the disk; Injection means: a reagent (biologically reactive substance)-containing layer arranged so that the liquid sample injected by the injection means is brought into contact with the liquid sample by being flowed toward the circumference by centrifugal force generated by rotation of the disk. A rotatable analytical disk is provided that includes: a gas permeable layer; an ambient air seal that protects the gas permeable layer from the ambient air; and an optical ellipsoid having an active surface capable of retaining a gas within the ambient air seal. Ru. Furthermore,
According to a preferred embodiment of the present invention, the rotatable analysis disk; means for rotating the disk; means for supplying a liquid sample to the liquid sample injection means in the center of the disk; means for measuring a surface plasmon phenomenon; A liquid sample analysis device including control means for each means is provided. Next, these preferred embodiments will be explained with reference to the attached drawings. As shown in FIGS. 3 and 4, the disk 21 is divided into a plurality of inspection sections 23 radially from the center 22 toward the circumference. Each test section 23 is separated from each other by a separation wall 24, and each test section 23 can independently analyze the same and/or different specific substances. Each test section 23 has a sample injection port 25 in the center for introducing a liquid sample into the system, and a discharge port 26 in the circumference for removing waste liquid after the reaction is completed. The central portion 22 is provided with a mounting means (not shown) for mounting on the rotation means of the analyzer (see Figure 1), which will be described later. Each compartment 23 is surrounded by a top wall 27, a bottom wall (disc main body) 28, and a side wall 29, and a sample receiving chamber 30 is located in the center of the disc.
It is equipped with As shown in FIGS. 4 and 5, in a wide area extending from the center to the circumference of the disk, the upper wall 2
A passage 31 extends below the sample receiving chamber 30 to the outlet 26. The passage 31 includes an enzyme membrane 32 and a gas permeable membrane 3.
It is in contact with the gas inspection room 34 via 3. The enzyme membrane 32 can also be filled within the passageway 31, as shown in FIG. An optically active body 35 (in the illustrated example, an active surface made of a resin surface roughened with a sand bath coated with a metal such as silver) is provided in the upper part of the gas inspection chamber 34 in contact with the gas permeable membrane 33. The optically active body 35 may be provided on the entire surface of the gas permeable film 33, or may be provided only on a portion thereof. The change in the amount of gas on the active surface of the optically active body 35 can be achieved by irradiating the active surface with an incident beam (e.g. laser light 36) from a suitable light source (not shown, e.g. a laser diode) and generating a surface plasmon phenomenon. Measurements can be made from the reflected beam 37 with suitable measuring means (not shown).
6 and the reflected beam 37, it must be able to transmit them. As shown in FIGS. 7 and 8, a sample passage 31 is provided at the bottom of the disk, a gas test chamber 34 is provided at the top of the disk, and an enzyme membrane 32 and a gas permeable membrane 33 are placed in the gas test chamber 34. The surface plasmon phenomenon can also be measured from above the disk by providing it at the bottom and/or side of the disk.

A filter layer 38 may be provided between the sample receiving chamber 30 and the passage 31 to remove impurities in the liquid sample. In addition to what is shown in FIG. 3, the test zones 23 provided on the disk may be separated by relatively wide isolation areas 41, for example as shown in FIG. It may be bent continuously or intermittently. Note that the inspection section (inspection section) may be provided directly and integrally on the disk, or may be provided by adhesive.

The material of the disk is not particularly limited, but may be made of plastic such as polycarbonate, polystyrene, polyacrylate or polymethacrylate, or glass. One embodiment of the preferred analyzer of the present invention is shown in Figure 10.
The disk 21 is detachably attached to rotating means 51 at its center. The rotating means 51 may be a known one, and may be 30
It is preferable to use one that can stably rotate up to about 0.000 rpm. The supply means 52 supplies the required amount of liquid sample to the sample injection port 25 provided at the center of the disk 21 via the supply port 53. As the supply 1,000 stages 52,
Usually, a microprobe with a position control mechanism is used. A waste liquid treatment means 56 consisting of a waste liquid storage section 54 and a waste liquid pipe 55 is provided outside the circumference of the disk 21. Furthermore, a measuring means 57 consisting of a suitable light source and a detector is provided outside the circumference of the disk 21. This measuring means 5
7 is provided at the top or bottom of the disk, depending on the location of the active surface within the disk. The rotation means 5l, the supply means 52 and the measurement means 57 are organically connected and controlled by the control means 58, so that the process from sample supply to measurement can be automated quickly and with high accuracy. Next, in order to carry out the enzyme method of the present invention using the above-mentioned analyzer, first, a liquid sample is introduced from the supply means 52 into the sample injection port 25 of the disk. Subsequently, the disk is rotated by the rotating means 51, and the liquid sample is caused to flow by centrifugal force and brought into contact with the enzyme membrane. When a test substance that reacts with the enzyme in the enzyme membrane is present in the liquid sample, the enzymatic reaction progresses and gas production and/or consumption occurs. This change in gas amount is retained on the active surface of the optical structure through the gas permeable membrane. The amount of gas on this active surface is measured by measuring means 57 using the surface plasmon phenomenon. After the measurement is completed, supply means 5
2, the cleaning solution is introduced into the sample injection port 25, the disk is rotated again, and the cleaning solution is caused to flow by centrifugal force to wash the enzyme membrane. The cleaning liquid is flushed out by centrifugal force into the waste liquid storage section 54 and removed from the waste liquid pipe 55. Next, in order to carry out the enzyme immunoassay, first, the supply means 5
2, a mixture of a liquid sample and an enzyme-labeled antigen (in the case of the competitive method) or an antibody (in the case of the sandwich method) is introduced into the sample injection port 25 of the disk. Subsequently, the disk is rotated by the rotating means 51, and the mixture is caused to flow by centrifugal force and brought into contact with the antibody-supported membrane. After the time required for the antigen-antibody reaction has passed, add the washing solution from the sample injection port, and
The disk is rotated again and the washing solution is flowed by centrifugal force to wash the antibody-carrying membrane and remove the liquid sample and the enzyme-labeled antigen or antibody that are not bound to the antibody on the antibody-carrying membrane. Next, pour the labeled enzyme substrate-containing solution through the sample injection port,
Rotate the disk again and use centrifugal force to flow the substrate-containing solution and bring it into contact with the antibody-supported membrane. When the enzyme-labeled antigen or antibody binds to the antibody on the antibody-supported membrane, the enzymatic reaction proceeds and gas is produced and/or consumed. Measure this change in gas amount in the same way as above. After the measurement is completed, the washing liquid is introduced from the supply means 52 into the sample injection port 25, the disk is rotated again, and the washing liquid is flowed by centrifugal force to wash the antibody-supporting membrane. The cleaning liquid is flushed out by centrifugal force into the waste liquid storage section 54 and removed from the waste liquid pipe 55. [Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples, but these are not intended to limit the scope of the present invention. Using a disk having the device shown in Figures 7 and 8,
The enzymatic method of the present invention was carried out using the apparatus shown in FIG.

That is, a suitable surface treatment was applied to the surface of a disc plate made of polycarbonate, and a nylon nonwoven fabric to which glucose oxidase was immobilized was placed as the biologically reactive substance-containing layer 32. The entire apparatus was maintained at 37°C. Known concentrations (10, 50, 100, 200, 500 and 1
0 00 mg/m 1 ) glucose standard solution
The sample was diluted 0 times, and 200 Jil of the diluted solution was injected from the sample injection port 25, and the disk was rotated to contact the glucose oxidase fixed layer. The optical structure 35 is irradiated with laser light to detect changes in oxygen consumed by the enzymatic reaction.
It was determined by measuring the Raman absorption change due to 02 at 1 cm"', and a calibration curve was prepared. Next, 200 serum samples containing unknown concentration of glucose were prepared.
A similar measurement using μl revealed that the serum sample contained 105 mg/ml of glucose.

[Effects of the Invention] According to the present invention, by combining an enzyme method or an enzyme immunoassay with a surface plasmon phenomenon measurement method, a test component in a liquid sample can be detected quickly, accurately, and with high precision. It can be analyzed using a simple process. Furthermore, performing the above analysis on a rotatable disk not only allows for more efficient analysis, but also allows multiple components to be analyzed simultaneously, allowing for automation and serialization with small sample volumes. Excellent effects such as

[Brief explanation of the drawing]

FIG. 1 and FIG. 2 are explanatory diagrams schematically showing the principle of the present invention. FIG. 3 is a plan view of one embodiment of the disk used in the present invention, and FIG. 4 is a sectional view taken along the line IV-IV in FIG.
FIG. 5 is a sectional view taken along the line V-V in FIG. 4, and FIG. 6 is a sectional view similar to FIG. 5 of another embodiment of the inspection section of the disk used in the present invention. FIG. 7 is a sectional view of still another aspect of the inspection section of the disk used in the present invention, and FIG. 8 is a sectional view taken along the line VTII-VIII of FIG. FIG. 9 is a plan view of another embodiment of the disk used in the present invention. FIG. 10 is an explanatory diagram schematically showing the analyzer of the present invention. Biologically reactive substance-containing layer...3, 32; Gas permeable layer...4, 33;

Claims (1)

[Scope of Claims] (1) Changes in the amount of gas caused by bringing a liquid sample into contact with a reagent-containing layer can be detected by an optical method using a surface plasmon phenomenon through a gas-permeable layer adjacent to the reagent-containing layer. A method for analyzing a liquid sample, characterized in that measurement is performed using a detection means. (2) Bringing a liquid sample into contact with the enzyme- or substrate-containing layer, and optically detecting the change in gas amount due to the enzyme reaction using the surface plasmon phenomenon through a gas-permeable layer adjacent to the enzyme- or substrate-containing layer. A method for analyzing a liquid sample, characterized in that measurement is performed by a method. (3) contacting a mixture of an enzyme-labeled first immunoreactive substance and a liquid sample with a layer containing a second immunoreactive substance; The reactive substance and the liquid sample are removed, a labeled enzyme substrate is added, and the change in gas amount due to the enzyme reaction is transferred to surface plasmon via the gas permeable layer adjacent to the second immunoreactive substance-containing layer. A method for analyzing a liquid sample, characterized by quantitatively measuring an immunoreactive substance using an optical detection means that utilizes a phenomenon. (4) A reaction between a liquid sample and a reagent is carried out on a rotatable disk, the surface plasmon active region supported on the disk is irradiated with light, and the liquid is detected by an optical detection means that utilizes the surface plasmon phenomenon. The method according to any one of claims 1 to 3, wherein the content or activity amount of a specific component in a sample is quantified. (5) Claims 1 to 3, wherein the optical detection means is Raman absorption.
4. The method according to any one of 4. (6) Liquid sample injection means; Reagent-containing layer arranged to be in contact with the liquid sample injected by the injection means; Gas permeable layer; Surface plasmon active region; Irradiating light to the surface plasmon active region. and an optical detection means that utilizes a surface plasmon phenomenon on the active surface. (7) The device according to claim 6, wherein the reagent-containing layer, the gas-permeable layer, and the surface plasmon active region are arranged on a rotatable disk.