JPH0514227B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention belongs to the technical field of clinical testing devices that measure various components in samples collected from living organisms.

[Technical background of the invention and its problems]

One method of clinical chemical analysis is the dry chemistry method. In the dry chemistry method, a sample is dropped onto paper impregnated with a reagent or a dry film coated with the reagent, and after the reagent and sample react,
A coloring reagent is applied to the film or paper portion onto which the sample has been dropped to develop a color, and the components in the sample are analyzed by measuring the absorbance.

However, this dry chemistry method
After the reaction between the reagent and the sample, a coloring mechanism is required to develop color in the reaction area, and the background must be suppressed in order to detect a specific wavelength in the colored area, and the background must be suppressed to detect the specific wavelength in the colored area. There is a problem in that the device becomes complicated because it requires an optical system.

In addition, in clinical chemistry analysis, as analytical methods that apply enzymes, there are a solution method in which the enzyme is used in a solution state and an immobilized enzyme method in which the enzyme is bound to an insoluble carrier and used repeatedly.

However, the solution method has the disadvantage of a short enzyme life, and the immobilized enzyme method does not have as much economic merit as originally expected, and the enzyme life is shorter than that of the solution method. Although it is long, it still has the drawback of being insufficient.

Furthermore, in clinical chemistry analysis, immunoassay methods include radioimmunoassay and enzyme immunoassay, but both require a long time, ranging from several hours to several tens of hours, before sufficient detection is possible. There are drawbacks.

[Purpose of the invention]

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a sample testing unit with a simple configuration that can easily and quickly quantify a specific substance in a liquid sample.

[Summary of the invention]

The specimen testing unit according to the present invention for achieving the above object is composed of a reagent layer in which a reagent is immobilized and held, and a gel, which is superimposed on the reagent layer and diffuses the sample at a uniform concentration. The method includes a developing layer that is transferred to the reagent layer, and a detection means that electrochemically detects a reaction product between the sample diffused from the developing layer and the reagent in the reagent layer, and dropping the sample onto the developing layer. The components of the sample are measured electrochemically.

[Embodiments of the invention]

An embodiment of the present invention will be described with reference to the drawings. FIG. 1 is an explanatory diagram showing an apparatus according to an embodiment of the present invention.

In FIG. 1, 3 is a reagent layer formed by holding a reagent on a carrier, and is formed on a reaction unit substrate 4 made of a poor conductor. On the upper surface of the reagent layer 3, a spreading layer 1 for uniformly spreading a sample of a predetermined concentration is superimposed via a diaphragm 2 such as a semipermeable membrane provided as necessary. An electrode insertion hole 5 is opened, for example, in the center of the reaction unit substrate 4, and an electrode part 6 can be attached to the electrode insertion hole 5. When the electrode part 6 is attached to the electrode insertion hole 5, the electrode protruding from the electrode part 6 is inserted into the reagent layer 3, so that the reaction products generated in the reagent layer 3 can be electrochemically detected. It's getting old. A lead wire 7 drawn out from the electrode section 6 is connected to an amplifier 8, and is configured to amplify the detection current output from the electrode section 6. The detected current is further output to a calculation unit 10 via a signal cable 9, and the calculation unit 10 calculates the concentration of a specific component in the sample.

The sample testing unit will be described in further detail.

The reagent layer 3, development layer 1, etc. can be configured as follows depending on the sample to be analyzed.

A case will be described in which a substrate component or an enzyme component in a sample is analyzed using the sample testing unit.

The carrier in the reagent layer 3 may be any carrier as long as it can hold or immobilize the enzyme or substrate through physical adsorption or chemical bonding, such as polyacrylamide, polyurethane, polyvinyl alcohol, polyvinylpyrrolidone. Gels of synthetic polymers with polar groups such as, gels of polysaccharides such as agarose, gels of proteins such as collagen and gelatin, gels of metal hydroxides such as aluminum hydroxide and titanium hydroxide, and ion exchange resins. ,
Examples include bonded supports such as porous glass beads, plastic membranes, zeolites, clays such as montmorillonite, etc., which may or may not have polar groups such as amide groups on their surfaces due to surface treatment. Enzymes or substrates are entrapped or immobilized on these carriers by physical adsorption or chemical bonding.

When analyzing a substrate component in a sample, the reagent in the reagent layer 3 is an enzyme that specifically enzymatically reacts with the substrate in the sample and can electrochemically analyze the enzymatic reaction product, or such an enzyme. Examples include microorganisms containing. The enzymes include oxidoreductases such as oxalate oxidase, pyruvate oxidase, lactate oxidase, D-amino acid oxidase, L-amino acid oxidase,
Monoamine oxidase, glucose oxidase, xanthine oxidase, diamine oxidase, alcohol oxidase, uricase, galactose oxidase, acyl coenzyme A
Oxidase, cholesterol oxidase, sarcosine oxidase, peroxidase, catalase, lactate dehydrogenase (LDH), glycerol-3-phosphate dehydrogenase, 3-hydroxybutyrate dehydrogenase, 3α steroid dehydrogenase, glycose-6-phosphate dehydrogenase, glucose dehydrogenase , malate dehydrogenase, isocitrate dehydrogenase, hydrolytic enzymes such as glutamate decarboxylase, aspartase, adenosine deaminase, nucleosidase, cytosine deaminase, creatine amidinohydrolase, urease, acetoacetate decarboxylase, alkaline phosphatase, acid phosphatase, Pyruvate decarboxylase, urokinase, phospholipase C, lipase, carboxypeptidase, leucine aminopeptidase, amylase,
Cholinesterase, aldolase, plasmin, trypsin, ribonuclease, deoxyribonuclease, protease, glucoamylase, transferase such as pyruvate kinase, myokinase, acyl coenzyme A synthetase, ornithine carbamyl transferase,
Examples include creatine kinase, aspartate aminotransferase, alanine aminotransferase, and isomerases such as glutamate racemase, alanine racemase, triosephosphate isomerase, and the like. In addition, when analyzing an enzyme component in a sample as a reagent in the reagent layer 3, a substrate that specifically enzymatically reacts with the above-mentioned enzyme, such as L-aspartic acid, L-alanine, α-
Ketoglutaric acid, pyruvic acid, L-lactic acid, L-
Leucinamide, glucose-6-phosphate, starch, acetylcholine, creatine, glutathione, ethanolamine, β-hydroxybutyric acid, ornithine, fructose bisphosphate, triglyceride, phosphoenolpyruvate, isocitric acid, malic acid, plasminogen , dopamine, ascorbic acid, protein, polynucleotide and the like.

In addition, examples of microorganisms containing the enzyme include Escherichia coli, Bacillus subtilis, Pseudomonas brevibacterium, Corynebacterium serratia,
Examples include Satucharomyces hansenula, Cryptocotrus canida, Aspergillus penicillium, Mucor, Rhizopus, Streptomyces, Actinomyces, and the like.

In preparing the reagent layer 3, the enzyme may be immobilized on the carrier or encased in a conventional manner. The above conventional method is described in books such as "Immobilized Enzymes" (edited by Ichiro Chibata, Kodansha, 1975) and Methods in Enzymology Vol. 34 (edited by WB Jacoby et al., Academic Press 1974). When the enzyme is bound to an insoluble carrier such as glass beads or cellulose, it is desirable to use a suitable binder or gel so that the immobilized enzyme settles on the reaction unit substrate.

The spreading layer 1 diffuses the sample so that the sample dropped on the upper surface of the spreading layer 1 can be transferred to the reagent layer 3 at a uniform concentration. gel can be used. By superimposing such a gel on the reagent layer, the dropped sample is also diffused in the direction in which the reagent layer spreads, so that the sample can be transferred to the entire reagent layer at a uniform concentration.

Electrochemical means can be used as the electrode part 6, for example, an ion electrode is used when the reaction product in the reagent layer 3 is an ion, and a hydrogen peroxide electrode is used when the reaction product is hydrogen peroxide. can be used.

The specimen testing unit shown in FIG. 1 is one embodiment of the apparatus, and may be modified as appropriate within the scope of the invention.

For example, instead of the electrode section 6 shown in FIG.
As shown in FIGS. 2A and 2B, a pair of electrodes 11 are arranged on the reaction unit substrate 4, and the electrodes 11 and the connection part 13 provided at the end of the reaction unit substrate 4 are electrically connected using a lead wire 12. The detection means may be configured such that the electrode 11 is embedded in the reagent layer 3 by connecting it to the reaction unit substrate 4 and forming the reagent layer 3 on the reaction unit substrate 4.

Using the sample testing unit (the apparatus shown in FIG. 1) configured as described above, the substrate component or enzyme component in a sample can be analyzed in the following manner.

First, when a sample, such as a blood sample, is dropped onto the surface of the developing layer 1, the blood sample diffuses in the developing layer 1 and reaches the reagent layer 3 with a uniform concentration. After reaching the substrate component (or enzyme component) in the blood sample and the enzyme (or substrate) retained in the reagent layer 3
triggers a specific enzymatic reaction. Then,
A reaction product is produced by the enzymatic reaction, and the reaction product is electrochemically quantified by the electrode unit 6 to determine the substrate component (or enzyme component) in the blood sample.
can be quantified.

Next, a case will be described in which an antigen or antibody in a sample is analyzed by the sample testing unit.

The carrier in the reagent layer 3 can be the same carrier used when the substrate component or enzyme component in the sample is analyzed by the sample testing unit.

When analyzing an antigen (or antibody) in a sample, the reagent in the reagent layer 3 binds the antibody (or antigen) to the cell membrane, which undergoes cytolytic action due to complement activation, and accommodates the enzyme (or substrate) inside. Examples thereof include microcapsules and immunoassay reagents containing a substrate (or enzyme) that specifically reacts with the enzyme (or substrate) contained within the microcapsules.

As microcapsules capable of binding antibodies (or antigens) and accommodating enzymes (or substrates), red blood cells of animals such as sheep can be suitably used. Note that even if red blood cells of other animals or animal cells other than red blood cells can be used as microorganisms in this invention, as long as they can bind antibodies (or antigens) to the cell membrane and contain enzymes (or substrates) within the cells. Can be used as capsules. Furthermore, liposomes can also be used as artificial membranes.

The antibody (or antigen) that binds to the cell membrane is appropriately selected to induce a specific antigen-antibody reaction with the antigen (or antibody) in the sample. For example, when the antigen in the sample is α-fetoprotein, an α-fetoprotein antibody can be used.

Further, examples of enzymes to be accommodated in the cells include the above-mentioned oxidoreductases, hydrolases, transferases, isomerases, and mixtures of the various enzymes mentioned above. Instead of accommodating the enzymes in the cells, a substrate that specifically reacts with the various enzymes described above may be accommodated. However, it is preferable that the substrate accommodated in the cell be a substance that does not easily permeate the cell membrane; for example, when the cell is a red blood cell, liposome, etc., chloride ions, glycerol, etc.
Examples include hydrophobic substances such as phospholipids, substances that can be permeated by active transport through the red blood cell membrane such as glucose, and amino acids.

The amount of enzyme (or substrate) accommodated in microcapsules can be freely adjusted during microcapsule preparation (Mitsuru Yoshizawa: Biochemistry 53 (9), 1066
(1981)). Therefore, the concentration of the enzyme or substrate contained in the microcapsules can be adjusted as appropriate depending on the concentration range of the antigen (or antibody) to be measured. Since the amount of intracellular enzyme (or substrate) is usually in large excess of the amount of antibody (or antigen), immunoassay measurements using the sample testing unit of the present invention can be carried out in a short period of time.

Cells, such as sheep red blood cells, in which an antibody such as an α-fetoprotein antibody is bound to the cell membrane and an enzyme such as glucose oxidase is housed within the red blood cells can be prepared, for example, as follows.

First, when α-fetoprotein is injected into animals such as rabbits, goats, mice, rats, etc. in a conventional manner, these animals become sensitized and produce α-fetoprotein in their bodies.
- Fetoprotein antibodies are produced. After an appropriate period of time has elapsed after the injection, a predetermined amount of blood is collected from the animal, and the supernatant of the blood is separated to obtain antiserum containing α-fetoprotein antibodies. Note that the antiserum may be further purified in order to improve the specificity of the antigen-antibody reaction. On the other hand, a predetermined amount of blood is collected from another animal, such as a sheep, and purified, and an isotonic solution, such as a sheep, is purified.
Buffer solution containing salt at a concentration of 0.15M (PH7)
An isotonic solution in which red blood cells are suspended is obtained by mixing with Since the red blood cells suspended in this isotonic solution contain cell fluid, mitochondria, etc., the cell fluid, etc. are expelled from the red blood cells using the standard dialysis method, and oxygen is returned to the red blood cells. Contains glucoamylase. The amount of glucoamylase to be accommodated can be appropriately determined depending on the needs of the immunoassay method described below. Next, an isotonic solution in which sheep red blood cells were suspended and the above α-
When mixed with an antiserum containing fetoprotein antibodies, the α-fetoprotein antibodies are adsorbed onto the cell membranes of sheep red blood cells, resulting in an isotonic solution with the red blood cells binding the α-fetoprotein antibodies to the cell membranes. . In this case, if the red blood cell membrane has difficulty adsorbing antibodies, antigens (or antibodies) can be attached to the membrane surface by treatment with a divalent reagent such as glutaraldehyde or succinic anhydride, or a peptide reagent such as Udward's reagent. can be combined.

The substrate (or enzyme) coexisting with the cells in the immunoassay reagent is the aforementioned substrate (or enzyme) that specifically enzymatically reacts with the enzyme (or substrate) accommodated in the cells. be able to.

To prepare the reagent layer 3, microcapsules can be encapsulated, for example, by a method using agar gel, which is commonly carried out in immunoassays. The method is described in detail in Introduction to Immunochemistry Experiments (Nao Matsuhashi et al., Gakkai Publishing Center (1981)) and Clinical Practice and Virology (Vol. 9, p. 479 (1981), Seiya Sato et al.).

As the developing layer 1, it is possible to use the same developing layer that is used when the substrate component or enzyme component in the sample is analyzed by the sample testing unit.

Note that the complement that dissolves cell membranes can be one that is contained in the blood of an animal; for example, guinea pig serum can be used as it is as a complement-containing liquid. Further, the complement may be added to either the reagent layer 3 or the developing layer 1 in advance, or may be added later as a secondary reagent.

As the electrode section 6, the same electrode as used when analyzing the substrate component or enzyme component in the sample with the sample testing unit can be used.

When the sample testing unit having the above configuration is used, antigens (or antibodies) in a sample can be quantified in the following manner.

First, when a sample, such as a blood sample, is dropped onto the surface of the developing layer 1, the blood sample diffuses in the developing layer 1 and reaches the reagent layer 3 with a uniform concentration. After reaching the antigen (or antibody) in the blood sample and reagent layer 3
Antibodies (or antigens) bound to the membrane surface of cells retained in the membrane induce a specific antigen-antibody reaction. Then, the complement that has been transferred with the blood sample from the spreading layer 1 or added to the reagent layer 3 is activated by the antigen-antibody reaction, and the cell membrane is dissolved by the cytolytic action of complement activity. The enzyme (or substrate) contained within the cell is released outside the cell. Enzyme (or substrate) released and substrate (or enzyme) in reagent
undergoes an enzymatic reaction in the reagent layer 3 to obtain a reaction product. Next, this reaction product can be electrochemically quantified using the electrode section 6. Note that the blood sample may be serum, plasma, or other whole blood.

The following examples further illustrate this invention.

Experimental Example 1 In this experimental example, glucose (substrate component) in a blood sample is quantified using the sample testing unit of the present invention.

By smoothly casting 0.1 M phosphate buffer (PH6.5) containing agarose (22 mg/ml) and Aspergillus-derived glucose oxidase (40 μg/ml) onto a plastic reaction unit substrate, and solidifying by cooling. A reagent layer was formed.
Next, 0.1 M phosphate buffer (PH6.5) containing agarose (22 mg/ml) was further layered on this reagent layer and solidified to form a spreading layer. In this case, the reaction unit substrate has an electrode insertion hole of a predetermined size, and a thin polyvinyl chloride film is pasted on the reaction unit substrate, and the electrode is inserted during the formation of the reagent layer. The pores are designed to prevent phosphate buffer from leaking out.
An electrode part is fitted into the electrode insertion hole, and the electrode is inserted into the reagent layer. Next, place about 10μ on the spreading layer.
serum was added dropwise. The serum diffuses through the spreading layer,
The concentration reached a certain level and moved to the reagent layer, where the following enzymatic reaction proceeded.

Glucose + O ₂ + H ₂ OGOD ---→ Gluconic acid + H ₂ O ₂ As a result of the reaction, H ₂ O ₂ was generated in the reagent layer. this
H ₂ O ₂ was detected using a hydrogen peroxide electrode in the electrode section.

FIG. 3 shows the relationship between the glucose concentration in the serum sample and the increase in the current output from the hydrogen peroxide electrode. As shown in FIG. 3, there is a linear relationship between the glucose concentration in the blood sample and the current increase value, and it is clear that the glucose in the blood sample can be quantified by measurement using a hydrogen peroxide electrode.
In addition, glucose was quantified using the same blood sample, and the quantification results are shown in Table 1. As shown in Table 1, quantitative determination using this sample testing unit can be performed with extremely high accuracy.

Table 1 Glucose 100mg/dl 250mg/dl Measurement 1 0.042 0.056 2 0.038 0.053 3 0.039 0.055 4 0.042 0.054 5 0.041 0.055 0.0403 0.0546 SD ±0.00179 0.00101 CV (%) 4.43 1.86 Experimental Example 2 This experimental example uses the specimen of this invention. This test unit is used to quantify α-fetoprotein (antigen, hereinafter sometimes abbreviated as α-FP) in a blood sample.

Agarose (12mg/ml), sensitized red blood cells (100μ
/ml; Contains 100U/ml glucoamylase,
α-FP antibody bound to the membrane surface), glucose oxidase (40 μg/ml), amylose (10 mg/ml), and complement (200 μ/ml) in phosphate buffered saline (PH6.0). A reagent layer was formed by smoothly casting onto a plastic reaction unit substrate and solidifying by cooling. A phosphate buffered saline solution (PH6.0) containing agarose (20 mg/ml) was further layered on this reagent layer and solidified to form a developing layer. In this case, the reaction unit substrate has an electrode insertion hole of a predetermined size, and a thin polyvinyl chloride film is pasted on the reaction unit substrate, and the electrode is inserted during the formation of the reagent layer. The pores are designed to prevent phosphate buffer from leaking out. An electrode part is fitted into the electrode insertion hole, and the electrode is inserted into the reagent layer. Next, approximately 10μ of serum was dropped onto the developing layer.

Next, approximately 10μ of serum was added onto the spreading layer. The serum diffused through the development layer, reached a constant concentration, and moved to the reagent layer, where antigen and antibody reactions proceeded. As a result, the membrane was destroyed by complement, and the glucoamylase contained within the blood cells (microcapsules) leaked out. The glucoamylase that comes out uses amylose as a substrate for the next reaction.

Amylose glucoamylase――――――――→ Glucose Glucose + O ₂ + H ₂ O Glucose oxidase――――――――――→ Gluconic acid + H ₂ O ₂ As a result of the reaction, H ₂ O ₂ is produced in the reagent layer. was generated. this
H ₂ O ₂ was detected using a hydrogen peroxide electrode in the electrode section. The reaction temperature was 37°C and the measurement time was 15 minutes.

A calibration curve is created in advance using a sample containing α-FP at a known concentration, and the calibration curve is used to calculate α-FP in the blood sample.
You can know the FP content.

Table 2 shows the results of quantifying α-FP using the same blood sample. As is clear from this, quantitative determination using this sample testing unit can be performed with extremely high accuracy.

Table 2 α-FP 48ng/ml 320ng/ml Measurement 1 0.025μA/〓 0.14μA/min 2 0.022 0.13 3 0.023 0.14 4 0.026 0.14 5 0.023 0.15 0.0238 0.14 SD ±0.00147 0.00632 CV(%) 6.17 4.51 [Effect of invention ] According to the present invention, it is possible to provide a sample testing unit with a simple configuration. In particular, since the components in the sample are quantified using electrochemical means based on the products of the reactions that have progressed in the reagent layer, not only is there no need for color development within the sample testing unit, but there is also a need for spectroscopic analysis. The need for a special layer for background cancellation, which is essential in the detection means, can also be eliminated. Moreover, various components in a sample can be accurately and quickly quantitatively analyzed depending on the type of reagent supported in the reagent layer. For example, if the enzyme (or substrate) is retained in the reagent layer, the substrate component (or enzyme component) in the sample can be accurately quantified. In addition, if the reagent layer carries an immunoassay reagent containing cells that bind antibodies (or substrates) to their cell membranes and contain enzymes (or substrates) inside, it is possible to dissolve the sample within several minutes to several tens of minutes. antigens (or antibodies) can be rapidly and quantitatively analyzed. Moreover, by superimposing the spreading layer made of gel on the reagent layer, even a minute sample can be transferred to the entire reagent layer at a uniform concentration, thereby improving measurement accuracy without wasting the sample. It is possible to improve the performance and speed up quantitative analysis.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing one embodiment of the device of the present invention, FIG. 2A is a sectional view showing another embodiment of the device of the invention, and FIG. 2B is a top view showing another embodiment of the device of the invention. 3 and 3 are graphs showing the relationship between the glucose concentration and the increase in the current output from the hydrogen peroxide electrode when glucose in a blood sample is quantified using an apparatus according to an embodiment of the present invention. . 1... Development layer, 3... Reagent layer, 6... Electrode section.

Claims (1)

[Scope of Claims] 1. A reagent layer in which a reagent is immobilized and held; a spreading layer formed of gel and superimposed on the reagent layer to diffuse a sample at a uniform concentration and transfer it to the reagent layer; It has a detection means that electrochemically detects a reaction product between the sample diffused from the developing layer and the reagent in the reagent layer, and the components of the sample are electrochemically detected by dropping the sample onto the developing layer. A specimen testing unit that measures 2. The sample testing unit according to claim 1, wherein the reagent includes an enzyme. 3. The sample testing unit according to claim 1, wherein the reagent contains a microorganism containing an enzyme. 4. Claim 4, characterized in that the reagent has a microcapsule that has an antibody or an antigen on its membrane that undergoes a lytic action due to complement activity, and contains an enzyme or a substrate that specifically reacts with the enzyme inside. The sample testing unit according to item 1.