JPH0443948A - Biochemical analysis and biochemical analysis sensor used in this method - Google Patents

Abstract

PURPOSE:To make simple and compact an analysis device and to speed up the measuring time by forming hydrogen peroxide by an enzyme reaction in which a material to be measured participates and detecting the material to be measured in accordance with the concn. thereof. CONSTITUTION:A gaseous hydrogen supply pipe 5 is connected to a constant- pressure hydrogen source and the inside of a sensor body 1 is maintained under a specified hydrogen pressure. The bottom end of the sensor is immersed into a sample soln. contg. the material to be measured and the material necessary for the prescribed enzyme reaction. The prescribed enzyme reaction on an enzyme immobilized film 6 takes place in this way and the hydrogen peroxide is formed. The formed hydrogen peroxide arrives at a measuring electrode 3 and is determined. The concn. of the material to be measured in the sample soln. is measured in accordance with the previously formed calibration curve from the quantity of the formed hydrogen peroxide determined in such a manner.

Description

DETAILED DESCRIPTION OF THE INVENTION [Purpose of the Invention (Field of Industrial Application) The present invention relates to a biochemical analysis method particularly useful in clinical testing, and a sensor for carrying out this analysis method.

More specifically, a biochemical analysis method involves generating hydrogen peroxide through an enzymatic reaction involving a substance to be measured, and detecting the substance to be measured based on the concentration of the generated hydrogen peroxide, and a sensor used for this analysis. related to.

(Prior Art) In biochemical analysis in clinical tests, various metabolites are measured. Examples of test items include total protein (TP),
Albumin (ALB), urea nitrogen (BUN), creatinine, uric acid (UA), electrolytes (Na", K",
C9-, Ca", Mg") glucose (GLU), neutral fat (T(1,:) liglyceride), total cholesterol (T-CHO), free cholesterol (F-CHO)
β-lipoprotein (β~L) Phospholipid, free fatty acid (FFA), inorganic phosphorus, iron, GOT, GPT, cholinesterase (Ch E), etc. Currently, various automatic biochemical analyzers capable of analyzing these test items are commercially available.

Among the above test items, for example, Na", Cjl-Ca"
For electrolytes such as, by measuring the potential of the solution to be measured using a sodium ion selective electrode, a chloride ion selective electrode, a calcium ion selective electrode, etc., respectively.
The concentration is determined based on the Nernst equation below.

E=a +:b 1 ogC However, E is the electrode potential, C is the concentration of the target ion, a and b are constants It is.

For measuring glucose, a glucose sensor has been developed in which a glucose oxidase-immobilized membrane is attached to a dissolved oxygen sensor. Also, for the measurement of urea nitrogen,
Urea nitrogen sensors have been developed in which a uricase-immobilized membrane is attached to an ammonia sensor.

However, for most other test items, a method is used in which a specific reagent is added to the substance to be measured to develop a color, and colorimetric determination is performed. For this reason, conventional automatic biochemical analyzers
A mechanism for adding several types of reagents to the substance to be measured and for performing colorimetric analysis is required. This has the disadvantage that the apparatus and operation are complicated, and furthermore, a large amount of expensive analytical reagents are required.

Also, in the case of the glucose sensor, the detection method is an amperometric method that detects the current flowing through the dissolved oxygen sensor. For this reason, it is difficult to apply potentiometry, which has recently come into use and can be integrated, to FET sensors as a basis for detection.

On the other hand, many of the above test items generate hydrogen peroxide through enzymatic reactions. Examples include glucose, neutral fat, total cholesterol, free cholesterol, phospholipids, free 111 fatty acids, inorganic phosphorus, cholinesterase, and the like. The clinical meaning of these test items is briefly explained as follows.

The normal value range for glucose (in the case of whole blood) is 50-10
0 D/d 11. Values lower than this correspond to deterioration of the pancreatic islets of Langerhans, parotid gland, liver disease, etc.

Also, values higher than normal correspond to diseases and illnesses such as hyperinsulinemia and liver disease.

The normal value range for neutral fat is 35-130 g/dN (adult, fasting). Values lower than this indicate endocrine disorders,
It deals with liver diseases, biliary tract diseases, intestinal diseases, etc. In addition, values higher than normal values correspond to diseases and disorders such as metabolic abnormalities and endocrine disorders.

The normal values for total cholesterol and free cholesterol are 130 to 250 mg/d II and 50 to 80 g/dl, respectively. Lower values correspond to endocrine disorders, liver diseases, etc. In addition, normal values and higher values correspond to diseases and illnesses such as metabolic abnormalities (diabetes, arteriosclerosis), liver disease, and biliary tract disease.

The normal value of phospholipids is 150-230 intestinal g/[1 (lecithin equivalent value). Values lower than this correspond to severe hepatic parenchymal disorder, severe anemia, leukemia, etc. Moreover, values higher than normal values correspond to diseases such as familial hyperlipoproteinemia, obstructive disease, jaundice, and hypothyroidism.

The normal value of free fatty acids is 176-586 μEq/j! It is. Values lower than this correspond to hypothyroidism, hypopituitarism, Agilan's disease, etc. Also, higher than normal values correspond to diseases and conditions such as diabetes, severe liver damage, and hyperthyroidism.

The normal value of inorganic phosphorus is 2.5-4.5 mg/d1
It is. Values lower than this correspond to hyperparathyroidism, vitamin D deficiency, etc. Values higher than normal correspond to diseases and conditions such as hypoparathyroidism, acromegaly, and chronic renal failure.

The normal value of cholinesterase is 3500-6000U
/II. Values lower than this are known to correspond to illnesses and illnesses such as liver disease and organophosphate poisoning.

As mentioned above, biochemical analysis of a substance to be measured which undergoes an enzymatic reaction to generate hydrogen peroxide occupies an important position in clinical testing as a whole. Therefore, if it is possible to test these measurement items using the same measuring device, it will be possible to test important items such as liver function, presence of abnormal blood sugar levels (diabetes detection), presence of arteriosclerosis, and thyroid function. It is hoped that it will become easier. In addition, if these items can be easily measured using a potentiometric hydrogen peroxide sensor or the like, there is a possibility that these inspection items can be integrated, and this would have great industrial value.

However, the method that has been carried out using conventional automatic biochemical analyzers involves oxidative condensation of 4-aminoantipyrine and phenol in the presence of the generated hydrogen peroxide and the enzyme peroxidase, and then colorimetrically quantifying the generated quinone pigment. The main methods were complicated methods such as

On the other hand, as mentioned above, sensors that quantify hydrogen peroxide have also been developed and are used to measure glucose, but these sensors use an enzyme (catalase) that acts on hydrogen peroxide and decomposes it to generate oxygen. This is what was used. Therefore, the determination of hydrogen peroxide is performed by quantifying the generated oxygen as dissolved oxygen, which requires a complicated amperometric method. As a result, it has been difficult to integrate the above inspection items and simplify measurements using such conventional hydrogen peroxide sensors.

Furthermore, in the conventional method using a hydrogen peroxide sensor equipped with a catalase-immobilized membrane, the response time is slower than in the method of directly measuring hydrogen peroxide concentration by changing the potential.
In addition, there is a problem that measurement accuracy decreases.

(Problems to be Solved by the Invention) As mentioned above, in the conventional biochemical analysis method, hydrogen peroxide is generated from the analyte through an enzymatic reaction, and the generated hydrogen peroxide is quantified to determine the analyte. Methods of analysis are known. However, there is no example of quantifying hydrogen peroxide using a potentiometric method.

That is, in glucose analysis, there are examples in which hydrogen peroxide is quantified using a sensor. However, in this case as well, complex amperometric quantitative methods are used. Furthermore, in most other examples, a method is used in which the generated hydrogen peroxide is quantified by a colorimetric method and the concentration of the substance to be measured is determined from this. Therefore, in each case, the operations and equipment have to be complicated, and in addition, it has been difficult to integrate these items even though the same hydrogen peroxide is to be quantified.

The present invention has been made in view of the above circumstances, and its first objective is to measure a substance to be measured that generates hydrogen peroxide through an enzymatic reaction by quantifying the hydrogen peroxide generated through the enzymatic reaction. The object of the present invention is to provide a method that can quantify hydrogen peroxide using an easy-to-operate potentiometric method, and can provide good measurement accuracy and quick response time.

A second object of the present invention is to provide a biochemical analysis sensor that enables implementation of the above method, particularly a sensor that can detect hydrogen peroxide concentration in a potentiometric manner by using a metal that has hydrogen storage properties. That's true.

More preferably, the present invention provides an analysis method and device that can perform biochemical analyzes of glucose, neutral fat, total cholesterol, free cholesterol, phospholipids, free fatty acids, inorganic phosphorus, cholinesterase, etc. using the same measuring device. That's true.

[Structure of the Invention] (Means and Effects for Solving the Problems) The first object of the present invention is to generate hydrogen peroxide from a substance to be measured through at least one step of enzymatic reaction, and to generate hydrogen peroxide. A biochemical analysis method for quantifying hydrogen peroxide and measuring the concentration of the substance to be measured based on the amount of hydrogen peroxide, wherein the hydrogen peroxide produced by the enzymatic reaction is contact with a measuring electrode made of a metal having hydrogen storage properties that outputs a voltage value corresponding to the amount of hydrogen peroxide, and quantifying hydrogen peroxide produced in the enzymatic reaction from the output voltage value of the measuring electrode. This is achieved by the following method.

The second a! of the present invention! 1m generates hydrogen peroxide from the substance to be measured through at least one step of enzymatic reaction, quantifies the generated hydrogen peroxide, and measures the concentration of the substance to be measured based on the amount of hydrogen peroxide. A sensor used in a biochemical analysis method, comprising a sensor body made of a hollow cylindrical body, the upper end of which is sealed, and at least a portion of the lower end having an opening, and a hydrogen storage device connected to a lead wire. a measurement electrode made of a metal thin film and provided to seal the lower end opening of the sensor body; a mechanism for maintaining hydrogen pressure inside the sensor body at a predetermined value; A biochemical analysis sensor characterized by having at least one type of enzyme immobilized thereon for generating hydrogen oxide, and comprising an enzyme immobilization membrane provided to cover the external surface of the measurement electrode. achieved by.

The metal thin film having hydrogen storage properties used in the present invention includes:
For example, simple metals such as palladium and platinum, or LaN
i1. Various materials can be used, such as Ti2Ni, T1Ni2°T1Ni, or alloys in which some of these metals are replaced with other metals. These metals and alloys can be rolled out as they are to form a thin plate, or these metals and alloys can be pulverized and then formed into a thin film. can be used.

The immobilized enzyme membrane used in the sensor of the present invention is one in which a desired enzyme is supported on an organic membrane such as a cellulose acetate membrane, a polyacrylonitrile membrane, or a vinyl chloride membrane by an inclusion method or a covalent bonding method. can be used.

Hereinafter, the present invention will be described in detail, including necessary operation explanations.

First, the biochemical analysis method of the present invention will be described. The feature of this method lies in the method for quantifying hydrogen peroxide produced from a substance to be measured by an enzymatic reaction. In this quantitative determination, one surface of the measurement electrode made of the metal thin film having hydrogen storage properties is kept under a predetermined hydrogen pressure atmosphere, and the other surface is brought into contact with the hydrogen peroxide-containing measurement solution. Measure the change in potential. Based on this potential change, the hydrogen peroxide concentration in the measurement liquid is determined. The quantitative principle is as follows.

One surface (first surface) of the hydrogen-absorbing metal empty measurement electrode
When a constant hydrogen pressure is applied to the surface, hydrogen is absorbed into the measuring electrode from this surface, and eventually reaches equilibrium with the applied hydrogen pressure. If this applied hydrogen pressure is left as it is, the other surface (second surface) of the measurement electrode also acts to keep the hydrogen pressure or hydrogen concentration constant. If the second surface is open, hydrogen will gradually escape from the open surface,
The hydrogen pressure on the second surface will reach equilibrium at a slightly reduced value. Furthermore, if the second surface is in a closed state, equilibrium is reached at a hydrogen pressure equivalent to the hydrogen pressure applied to the first surface.

Therefore, if an aqueous solution containing hydrogen peroxide is in contact with the second surface of the measurement electrode, the hydrogen peroxide and the hydrogen occluded in the measurement electrode will react to generate H2O. decrease the hydrogen pressure. This becomes clear when comparing the following reaction that occurs at the electrode interface in a basic solution.

H2O2+2e = 20 H-(+0.88V vs, NHE)
・"■2H20+26" 20 H-+ H2 (-0J3V vs, N) IE
) ・The reaction represented by the formula ■■ is a redox reaction between hydrogen peroxide and hydroxide ions, and the reaction represented by the formula ■ is a redox reaction between hydrogen, water, and hydroxide ions.

The value of the redox potential written together with the formula (■■) indicates that the following formula (■'), which is the reduction reaction of (2), and the following formula (■'), which is the oxidation reaction of (2), occur in a conjugate manner.

Reduction reaction H202+2 e 420 H--■'■ Oxidation reaction 20 H-+H2-2H20+ 2 e -■'As a result, the reaction of the following formula (2) occurs due to ■'+■'.

H2O2+2H(1/2 H2) →2H20...■ Due to this reaction (■), the hydrogen concentration on the second surface decreases.

On the other hand, there is a relationship expressed by the following equation (2) between the potential Eeq of the measurement electrode and the activity of hydrogen occluded in the electrode.

E, Qm Eo (RT/ 2 F) l
na 82 ”・■ However, R is the gas constant T is the absolute temperature F is the Faraday constant Eo is the potential when the activity of hydrogen (882) is 1, and is a constant.

Therefore, when the hydrogen at the second surface of the measuring electrode (ie, the hydrogen activity a H2) decreases, E, shifts in the positive direction according to equation 0. Therefore, the greater the amount of hydrogen peroxide, the more the potential shifts in the positive direction. Since there is a 1:1 relationship between the shift amount (ΔE) and the amount of hydrogen peroxide, E increases monotonically as the amount of hydrogen peroxide increases. As a result, the hydrogen peroxide concentration can be determined by creating a calibration curve showing the relationship between the hydrogen peroxide concentration and ΔE.

In the biochemical analysis method of the present invention, the substance to be measured is measured from the hydrogen peroxide concentration determined by the above method. In order for this to be possible, hydrogen peroxide generated from the substance to be measured by an enzymatic reaction must reach the second surface without fail. For this purpose, as in the sensor according to the present invention, an enzyme-immobilized membrane containing an enzyme necessary for generating hydrogen peroxide may be brought into close contact with the second surface. This ensures that the generated hydrogen peroxide reaches the second surface, and the potential of the metallic thin film changes depending on the amount. In this way, the amount of hydrogen peroxide to be quantified corresponds to the concentration of the substance to be measured, so by examining the potential change, it is possible to quantify the substance to be measured.

In addition, the above-mentioned formula ■ which is the basis of the measurement principle in the present invention
, ■ are all electrode reactions in a basic electrolyte solution. Therefore, in the method of the invention it is desirable to cover the second surface of the measuring electrode with a thin layer of an electrolyte solution, preferably a basic electrolyte solution, in order to allow the hydrogen peroxide formed to diffuse into this electrolyte solution. . To do this, the first surface of the measuring electrode may be kept under a predetermined hydrogen pressure atmosphere, and the second surface may be brought into contact with a hydrogen peroxide-containing measuring solution via a thin layer of electrolyte.

Furthermore, when carrying out the biochemical analysis method according to the present invention, it is preferable that the enzymatic reaction that generates hydrogen peroxide is carried out on the second surface of the measurement electrode. For this purpose, for example, an enzyme-immobilized membrane containing one or more types of enzymes necessary for the enzyme reaction may be placed in close contact with the second surface of the measurement electrode.

However, when using a thin layer of the electrolyte solution, the thin layer is interposed between the enzyme-immobilized membrane and the measurement electrode.

Furthermore, when a third substance is required in addition to the substance to be measured and the enzyme in the enzyme reaction that generates hydrogen peroxide, the third substance may be supplied in advance to the sample solution or the enzyme-immobilized membrane. put.

Next, the substances to be measured to which the biochemical analysis method of the present invention can be applied will be classified into several aspects and explained along with the enzyme reactions in each case. Note that all the enzyme reactions used below are known ones.

<A> Requiring only one type of enzyme (1) The enzyme that uses glucose is glucose oxidase. The enzymatic reaction in this case is expressed by the following formula.

D-glucose + 0□ + H20 → D-gluconic acid decaH2O2 (2) Uric acid In this case, the enzyme used in the reaction to produce hydrogen peroxide is uricase.

(3)? In this case, the enzyme used in the reaction to produce hydrogen peroxide is cholesterol oxidase.

<B> 2 [Those that require only enzymes of the same class or higher (
1) Phospholipid As an enzymatic reaction to produce hydrogen peroxide from phospholipid, a two-step reaction shown by the following formula can be used.

Phospholipid 1 H2o → Choline Choline + 02 + H20 → Betaine + H2O2 In the first step of the reaction, choline is produced by hydrolysis of phospholipids. This reaction requires phospholibase D as an enzyme.

In the second stage of the reaction, choline is oxidized to produce hydrogen peroxide. This reaction requires choline oxidase. Therefore, these two types of enzymes are necessary.

(2) Total cholesterol Total cholesterol is the sum of the amount of cholesterol esters and the amount of free cholesterol. In this measurement, cholesterol ester is hydrolyzed, and the free cholesterol produced thereby and the free cholesterol originally present are measured simultaneously.

Therefore, in this case, cholesterol hydrolase for hydrolyzing cholesterol esters and cholesterol oxidase necessary for the reaction of generating hydrogen peroxide from free cholesterol are required.

Note that although it is desirable to use cholesterol oxidase as an enzyme-immobilized membrane, cholesterol hydrolase may be added to the liquid to be measured in advance.

<C> Requiring an enzyme and a third substance (1) Inorganic phosphorus As the enzymatic reaction for producing hydrogen peroxide from inorganic phosphorus, a two-step reaction shown by the following formula can be used.

Inorganic phosphorus + inosine → hypoxanthine hypoxanthine + 02 → xanthine + H2O2 In the first step reaction, hypoxanthine is produced from inorganic phosphorus and inosine by the action of purine nucleoside phosphorylase.

In the second step, xanthine oxidase oxidizes hypoxanthine to produce hydrogen peroxide.

Therefore, in this case, the above two types of enzymes are required.

In addition, inosine is required as a third substance in the first step reaction. Inosine may be used by adding it to the solution to be measured in advance.

(2) Triglyceride To produce hydrogen peroxide from triglyceride, an enzymatic reaction consisting of the following three-step reaction can be used.

In the first step of the reaction, triglyceride is treated with lipoprotein lipase. As a result, triglyceride is hydrolyzed to produce glycerol.

In the second step, glycerol kinase is allowed to act on glycerol in the presence of ATP.

This phosphorylates glycerol to produce glycerol-3-phosphate.

In the third step, glycerol-3-phosphate oxidase acts on glycerol-3-phosphate.

This oxidizes glycerol-3-phosphate to produce hydrogen peroxide.

Therefore, in this case, in addition to the three enzymes mentioned above, ATP, which is consumed as an energy source in the second reaction, is required. This ATP may be added to the liquid to be measured in advance.

<D> A substance to be measured is an enzyme, a substrate for the enzyme is required as a third substance, and another enzyme is also required (1) Cholinesterase A reaction in which cholinesterase is involved and produces hydrogen peroxide. , a two-step reaction represented by the following formula can be used.

Benzoylcholine - Choline Choline debenzoate + O2 + H20 - Betaine + H2O2 The hydrolysis reaction of benzoylcholine in the first step is:
This is an enzymatic reaction involving the substance to be measured, cholinesterase. This reaction requires benzoylcholine as a substrate for cholinesterase. Benzoylcholine may be added in advance to the liquid to be measured together with the cholinesterase to be measured.

The second step is the oxidation of choline to produce hydrogen peroxide, which requires choline oxidase as an enzyme. This choline oxidase is preferably used as an enzyme-immobilized membrane.

<E> Those that require an enzyme, a third substance to activate the enzyme, and a third substance to drive the enzyme reaction.

(1) Free fatty acids i! As a reaction for producing hydrogen peroxide from i8 fatty acids, a two-step reaction shown by the following formula can be used. This reaction involves acyl-CoA synthetase and acyl-C
It is a coupled enzymatic reaction involving two enzymes, oA oxidase.

RCOO)l + ATP + CoA-acyl-Co
A+ AMP +PPI acyl-Co^+02 →2,3-transenol-CoA+ H202 However, in the above formula, ATP is adenosine triphosphate, and CoA is coenzyme A.

AMP is adenosine-phosphate, PPi is birophosphoric acid It is.

The first step reaction requires acyl-CoA synthetase as an enzyme. It also requires Mg2+ ions to activate acyl-CoA synthetase. In addition, ATP and coenzyme CoA are required as energy sources to drive this enzymatic reaction.

The second stage reaction requires acyl-CoA oxidase as an enzyme.

Among the above-mentioned necessary third substances, M g2+ ions are preferably added to the enzyme-immobilized membrane as sparingly soluble magnesium ions, together with the two necessary enzymes. Also, A
TP may be added to the liquid to be measured in advance.

The biochemical analysis sensor according to the present invention has the necessary configuration to carry out the analysis method described above. Its basic configuration is as described above. As a mechanism for maintaining the hydrogen pressure inside the sensor body at a predetermined value, a hydrogen gas supply source may be provided, for example, via a hydrogen gas supply pipe connected to the hollow interior. Further, a reference electrode may be used in addition to the measurement electrode, and as described above, a thin layer of the electrolyte solution may be interposed between the enzyme-immobilized membrane and the measurement electrode. A more detailed configuration will be explained in the following example.

As described above, according to the present invention, various substances to be measured that generate hydrogen peroxide can be measured. Moreover, in the present invention, since each item is detected based on the amount of hydrogen peroxide produced, it is easy to integrate.

(Example) Hereinafter, the present invention will be explained in more detail based on examples.

FIG. 1 is a sectional view showing a biochemical analysis sensor according to an embodiment of the present invention. In the figure, reference numeral 1 denotes a hollow cylindrical sensor body whose upper end is sealed and which has an opening 2 at its lower end. The opening 2 is sealed with a measuring electrode 3 made of a palladium-containing alloy thin film to which a lead wire 4 is connected. The sensor body 1 is provided with a hydrogen gas supply pipe 5 communicating with a hollow portion thereof. This pipe 5 is connected to a constant pressure hydrogen gas supply source (not shown) in order to maintain the hydrogen pressure inside the sensor body 1 at a predetermined value. Further, an immobilized enzyme membrane 6 on which an enzyme such as uricase is immobilized is provided to cover the entire external surface of the measurement electrode 3. The enzyme-immobilized membrane 6 also contains a third substance necessary for a predetermined enzyme reaction, if necessary.

When carrying out the analysis method of the present invention using the sensor shown in FIG. 1, the hydrogen gas supply pipe 5 is connected to a constant pressure hydrogen source, and the inside of the sensor body 1 is maintained at a constant hydrogen pressure.

In this state, the lower end of the sensor is immersed in a sample solution containing the substance to be measured and a substance necessary for a predetermined enzymatic reaction. As a result, a predetermined enzyme reaction occurs on the enzyme-immobilized membrane, and hydrogen peroxide is produced. The generated hydrogen peroxide reaches the measuring electrode 3 and is quantified as described above. The concentration of the substance to be measured in the sample solution is measured from the amount of hydrogen peroxide produced determined in this way, according to a calibration curve prepared in advance.

FIG. 2 is a sectional view showing a biochemical analysis sensor according to another embodiment of the present invention. In the figure, reference numeral 11 denotes a hollow cylindrical sensor body whose upper end is sealed and which has an opening 12 at its lower end. The opening 12 is sealed with a measurement electrode 13 made of a titanium-nickel hydrogen storage alloy thin film. A lead wire 15 is connected to the measurement electrode 13 via a current collector 14 . Further, the sensor body 11 is provided with a hydrogen gas supply pipe 16 that communicates with the hollow portion thereof. Furthermore, surrounding the entire external surface of the measuring electrode 13,
A cellulose acetate membrane 17 permeable to dissolved hydrogen peroxide is provided. Between the cellulose acetate membrane and the measurement electrode 13, a basic electrolyte thin layer of 0.5MK
A trishydroxymethylaminomethane-carboxylic acid mixture 18 to which Cfi has been added is stored. A reference electrode 19 is provided that is in contact with this basic electrolyte solution thin layer 18 and is insulated from other parts. A lead wire 20 extending outside is connected to this reference electrode. Further, an immobilized enzyme membrane 21 on which an enzyme such as glucose oxidase is immobilized is provided in close contact with the outside of the cellulose acetate membrane 17. The enzyme-immobilized membrane includes:
If necessary, a third substance necessary for a predetermined enzymatic reaction is also contained.

Even when the sensor shown in FIG. 2 is used, the biochemical analysis method of the present invention can be carried out in the same manner as when the sensor shown in FIG. 1 is used. In this case, the basic electrolyte solution thin layer 1
By providing the reference electrode 8 and the reference electrode 19, more accurate analysis can be performed.

FIG. 3 shows an example of a biochemical analysis system using the sensor of the present invention. In the figure, numeral 31 is a biochemical analysis sensor having the same configuration as shown in FIG. A flow cell 32 is provided that fixes the sensor 31 and allows the measurement liquid to flow so as to come into contact with the sensitive part of the sensor 31. 33 is a flow path inside the flow cell 32. On the other hand, a sample tube 34 containing a sample liquid to be analyzed
, a container 35 containing a calibration solution containing the third substance mentioned above.
, a container 36 for containing a diluent, and a reaction tank 37 for preparing a liquid to be measured. In the reaction tank 37, the pump 41
A liquid to be measured is prepared by stirring and mixing the diluting liquid supplied by the pump 40 and the sample liquid and/or calibration liquid supplied by the pump 40. In addition, calibration solution, dilution solution,
The liquid to be measured is fed through piping 39. Further, the pump 40 is equipped with a mechanism that allows selection of whether to aspirate the calibration liquid or the sample liquid.

The liquid to be measured prepared in the reaction tank 37 is supplied to the internal flow path 33 of the flow cell 32 through the piping 39 by driving the pump 42 . The liquid to be measured comes into contact with the sensitive part of the sensor 31 here. As a result, hydrogen peroxide is generated by the mechanism described above, and its amount is quantified. After the measurement is completed,
The liquid to be measured is discharged through the pipe 39 and is transferred to the waste liquid tank 38.
be accommodated in. The pump 42 also serves as a pump for discharging this waste liquid. Note that 43 is a recording and analysis mechanism that records and analyzes the output from the biochemical analysis sensor 31;
44 is a display mechanism that displays the analysis results. Also,
45 is a control mechanism that controls the entire measurement system.

According to the biochemical analysis system configured as described above, by replacing the sensor 31, the biochemical analysis of the present invention can be easily performed on various items.

At that time, in addition to the enzyme and the substance to be measured, enzyme substrates and A
Even when a third substance such as TP is required, it can be easily handled because it can be added to the sample liquid 34 by including it in the diluent 36.

FIG. 4 shows another example of a biochemical analysis system using the sensor of the present invention. In the figure, reference numeral 51 denotes a flow cell in which a plurality of biochemical analysis sensors of the present invention are fixed and a liquid to be measured is caused to flow so as to come into contact with the sensitive parts of the plurality of biochemical analysis sensors.

A flow path 52 is provided within the flow cell 51 in order to circulate the liquid to be measured in this manner.

The flow path 52 is provided with two inlets 53 and 54 for the liquid to be measured, first and second, and one outlet 55 for discharging the liquid to be measured after the measurement is completed. Two inlets 5
The respective liquids to be measured flowing in from 3.54 are designed to join together before the outlet 55. In order to prevent contamination between the liquids to be measured during this merging, the flow path 52 is sufficiently long.

The flow cell 51 includes a biochemical analysis sensor 6 according to the present invention.
1,62,63,64 are fixed. All of these sensors have the structure shown in FIG. Moreover, each of the sensors 61 to 62 is configured as a uric acid sensor, a glucose sensor, a phospholipid sensor, and a free cholesterol sensor.

These sensor groups are arranged along a flow path 52 connected to the first liquid to be measured inlet 53. The immobilized enzyme membranes include a uricase-immobilized membrane for the uric acid sensor 61, a glucose-immobilized membrane for the glucose sensor 62, a membrane immobilized with both phospholipase-D and choline oxidase, and a free cholesterol sensor for the phospholipid sensor 63. 64 uses a cholesterol oxidase-immobilized membrane, respectively.

The flow cell 51 also has a second liquid inlet 5 to be measured.
Along the flow path connected to 4, another biochemical analysis sensor 65
, 66, 67, 68, 69 are fixed. These sensors 65 to 69 also have the same structure as the biochemical analysis sensor shown in FIG. 2, and are configured as a free fatty acid sensor, a triglyceride sensor, a cholinesterase sensor, an inorganic phosphorus sensor, and a total cholesterol sensor, respectively. That is, the free fatty acid sensor 65 contains acyl-CoA.
Immobilize synthetase and acyl-CoA oxidase,
In addition, an enzyme-immobilized membrane containing a sparingly soluble magnesium salt is used. The triglyceride sensor 66 uses an enzyme-immobilized membrane on which lipoprotein lipase, glycerokinase, and glycerol-3-phosphate oxidase are immobilized. The cholinesterase sensor 67 uses an enzyme-immobilized membrane on which choline oxidase is immobilized. The inorganic phosphorus sensor 68 uses an immobilized enzyme membrane on which purine nucleoside phosphorylase and xanthine oxidase are immobilized. The total cholesterol sensor 69 uses an immobilized enzyme membrane on which cholesterol oxidase is immobilized.

70 is a first sample tube containing a sample liquid to be analyzed. This first sample tube contains a first sample liquid to be analyzed for uric acid, glucose, phospholipids, and free cholesterol. Generally, the first sample liquid is a body fluid such as serum, whole blood, or urine. A calibration liquid necessary for analyzing this first sample liquid is contained in a container 71. The diluted solution was prepared by adding each of the components to be measured at predetermined concentrations. Further, a diluent necessary for analyzing the first sample liquid is stored in the container 72. This dilution is
A buffer solution containing trishydroxymethylaminomethane and boric acid. 73 is a reaction tank for preparing a liquid to be measured by mixing a sample liquid, a dilution liquid, and/or a calibration liquid. In the reaction tank 73, 2. A first liquid to be measured is prepared by stirring and mixing the sample liquid and/or calibration liquid supplied by the pump 80 and the dilution liquid supplied by the pump 81. Note that the calibration solution, dilution solution, and sample solution are fed through piping 79. Further, the pump 80 is equipped with a mechanism that allows selection between suction of the calibration liquid and suction of the sample liquid.

On the other hand, the second sample tube 74 contains i! ! A second sample solution for analyzing fatty acids, triglycerides, cholinesterase, inorganic phosphorus, and total cholesterol is accommodated. This second sample solution is also generally serum, whole blood,
Body fluids such as urine. The calibration solution necessary for this analysis is in container 7.
It is housed in 5.

This calibration solution was prepared by adding each of the above measurement components at a predetermined concentration. Further, a diluent necessary for analyzing the second sample liquid is stored in the container 76.

In addition to trishydroxyaminomethane and boric acid, this diluted solution contains the third substances ATP, benzoylcholine, inosine, and cholesterol hydrolase at predetermined concentrations. 77 is a reaction tank for preparing a liquid to be measured by stirring the second sample liquid and/or calibration liquid and a diluting liquid. In this reaction tank 77, the sample liquid and/or calibration liquid supplied by the pump 82 and the pump 83
By stirring and mixing the diluent supplied by
A second liquid to be measured is prepared. Note that the calibration solution, dilution solution, and sample solution are fed through piping 79.

Further, the pump 82 is equipped with a mechanism that allows selection between suction of the calibration liquid and suction of the sample liquid.

The first liquid to be measured prepared in the reaction tank 73 and the reaction tank 77
The second liquid to be measured, which has been prepared by
supplied to Here, the first liquid to be measured is the sensor 61~
64, and the second liquid to be measured contacts the sensor 65~
Touch the sensitive part of 69. As a result, hydrogen peroxide is produced by an enzyme reaction corresponding to each sensor, and its amount is quantified. After the measurement is completed, the first and second liquids to be measured are discharged through the pipe 55 and stored in the waste liquid tank 78. The pump 84 also serves as a pump for discharging this waste liquid.

Note that 85 is a recording and analysis mechanism that records and analyzes the output from the biochemical analysis sensor, and 86 is a display mechanism that displays the analysis results. Further, 87 is a control mechanism that controls the entire measurement system.

According to the biochemical analysis system shown in FIG. 4, uric acid, glucose, phospholipids, free cholesterol, free fatty acids, triglycerides, cholinesterase, inorganic phosphorus, and total cholesterol can be measured simultaneously. Additionally, this biochemical analysis system is compact and has a simpler mechanism, unlike conventional analysis systems that mainly use colorimetry. Furthermore, when the concentration of each component in serum was measured using this analysis system, the quantification of each component was quickly performed within 20 minutes. In contrast, in conventional analysis systems, it took about two hours to complete the measurement because it was necessary to add the reagent to the liquid to be measured and then allow it to react until color was developed. Furthermore, since the apparatus of the present invention does not require a reagent for color development, analysis costs can be reduced.

In the above embodiments and measurement system examples, in order to keep the hydrogen pressure or hydrogen concentration on the surface of the measurement electrode constant during non-measurement periods, a means for supplying hydrogen from the outside through a pipe is used. However, as shown in the embodiment of FIG.
The measurement electrode absorbs a predetermined amount of hydrogen relative to the reference electrode,
It is also possible to apply a predetermined bias voltage so that the hydrogen concentration on the surface of the measurement electrode becomes a predetermined value.

In FIG. 5, 101 is a sensor body 103 whose upper end is sealed and whose interior is hollow and has an opening 102 at its lower end.
is a measurement electrode made of a thin film of palladium-containing alloy connected to a lead wire 104 that seals the opening. In addition to these, a reference electrode 105 is provided in this embodiment. The reference electrode is used to measure the potential of the measurement electrode 103 and to keep the hydrogen concentration on the surface of the measurement electrode 103 at a predetermined value by applying a predetermined bias to the metallic thin film when not measuring.

Reference numeral 106 denotes an immobilized enzyme membrane on which uricase is immobilized, covering the entire exterior of the metal thin membrane. Reference numeral 107 denotes a control mechanism that measures the potential of the measurement electrode 103 and performs controls such as applying a predetermined bias to the measurement electrode 103 as necessary.

The embodiment shown in FIG. 5 has the advantage that by applying a bias to the measurement electrode 103, the hydrogen atmosphere on the surface of the measurement electrode, that is, the hydrogen concentration, can be maintained at a constant value when not measuring, and the measurement system can be made compact. be. Therefore, it is particularly effective for small-sized measurement systems and measurement systems that integrate multiple items.

FIG. 6 shows F in the biochemical analysis sensor according to the present invention.
1 shows a schematic cross-sectional view of an embodiment based on an ET sensor; FIG.

In the figure, 111 is a silicon substrate, 112,
113 are a source and a drain, respectively. 114 is S
The iO□ layer 115 is a Si3N4 layer. Said Si, N
A measurement electrode 116 made of a hydrogen-absorbing metal thin film is formed as a gate electrode on the fourth layer 115. A lead portion 117 extends outward from the measurement electrode. 118 is an immobilized enzyme membrane that covers the measurement electrode 116.

The FET structure sensor shown in FIG. 6 has the advantage that several items can be easily integrated. The control mechanism 10 shown in FIG. 5 controls both the sensor shown in FIG. 6 and the reference electrode.
7 and the FET sensor drive mechanism can be used together. Thereby, it is possible to measure the potential of the measuring electrode and to maintain a constant hydrogen concentration on the surface of the measuring electrode by applying a predetermined bias to the measuring electrode when not measuring.

As shown in Fig. 7, the FET sensor as described above is
It is also possible to adopt a configuration having a plurality of detection units. That is,
Figure 7 shows an FET sensor in which three types of immobilized enzyme membranes, glucose oxidase immobilized membrane, uricase immobilized membrane, and cholesterol oxidase immobilized membrane, are provided on three independent gates, viewed from above the gate part. FIG. In the figure, 121 is the FET sensor body. Further, 122, 123, and 124 are a glucose detection section, a uric acid detection section, and a free cholesterol detection section, each of which has a glucose oxidase-immobilized membrane, a uricase-immobilized membrane, and a cholesterol oxidase-immobilized membrane, respectively.

Next, examples of specific biochemical analyzes performed according to the method of the present invention will be described.

Example 1 (Analysis of glucose) By using a sputtering method, a film with a thickness of 2
A μ-T1Ni alloy thin film was formed.

The surface of this alloy thin film was coated with an enzyme-immobilized film on which glucose oxidase was immobilized. Using this as a detection electrode, a biochemical analysis sensor shown in FIG. 2 was fabricated.

The sensor prepared above was set in the biochemical analysis system described in FIG. 3, and a standard solution with a known glucose concentration was measured in order to create a calibration curve. At that time, the difference (ΔE) between the measured electrode potential before the enzymatic reaction and the measured electrode potential when the reaction progressed and reached a steady value was measured. When ΔE was plotted against the logarithm of each glucose concentration (Cg), the relationship shown in FIG. 8 was obtained.

The results in Figure 8 indicate that there is an approximately linear relationship between the logarithm of Cg and ΔE in a range that includes the normal range of glucose concentration in human blood (50 to 100 mg/d II). ing. Therefore, it can be seen that the method of the present invention is effective for measuring glucose concentration.

Example 2 (Analysis of Phospholipids) An enzyme-immobilized membrane in which two types of enzymes, phospholipase D and choline oxidase, were immobilized was used. The biochemical analysis sensor shown in FIG. 2 was produced in the same manner as in Example 1 except for the above.

The sensor prepared above was set in the biochemical analysis system described in FIG. 3, and a standard solution with a known phospholipid concentration was measured in order to create a calibration curve.

At that time, the difference (ΔE) between the measured electrode potential before the enzymatic reaction and the measured electrode potential when the reaction progresses and reaches a steady value.
ΔE for the logarithm of each phospholipid concentration (Cp)
was plotted. As a result, the relationship shown in FIG. 8 was obtained.

The results shown in Figure 8 indicate that there is an approximately linear relationship between the logarithm of Cp and ΔE in a range that includes the normal range of phospholipid concentration in human blood (150 to 230 g/dl). It shows. Therefore, it can be seen that the method of the present invention is effective for measuring glucose concentration.

Example 3 (Analysis of Inorganic Phosphorus) An enzyme-immobilized membrane on which two types of enzymes, purine nucleoside phosphorylase and xanthine oxidase, were immobilized was used. The biochemical analysis sensor shown in FIG. 2 was produced in the same manner as in Example 1 except for the above.

The sensor prepared above was set in the biochemical analysis system described in FIG. 3, and in order to create a calibration curve, measurements were performed using a standard solution with a known phospholipid concentration to which inosine had been added. At that time, the difference (Δ
E) was measured. As a result of plotting ΔE against the logarithm of each inorganic phosphorus concentration (Cip), the relationship shown in FIG. 9 was obtained.

The results in Figure 9 show that the range includes the normal range of inorganic phosphorus concentration in human blood (2.5 to 4.5 mg/d 1 ).
It is shown that there is a substantially linear relationship between the logarithm of C1p and ΔE. Therefore, it can be seen that the method of the present invention is effective for measuring inorganic phosphorus.

Example 4 (Analysis of cholinesterase) An enzyme-immobilized membrane on which choline oxidase was immobilized was used.

The biochemical analysis sensor shown in FIG. 2 was produced in the same manner as in Example 1 except for the above.

The sensor prepared above was set in the biochemical analysis system described in FIG. 3, and a standard solution with a known cholinesterase concentration was measured in order to create a calibration curve. Benzoylcholine, a substrate for cholinesterase, was added to this standard solution in advance. During measurement, the difference (ΔE) between the measured electrode potential before the enzymatic reaction and the measured electrode potential when the reaction progresses and reaches a steady value is measured, and ΔE is calculated based on the logarithm of the cholinesterase concentration (Cche). was plotted. As a result, the relationship shown in FIG. 10 was obtained.

The results in Figure 10 indicate the normal range of cholinesterase concentration in human blood (3500-6000 U/d#)
It is shown that there is a substantially linear relationship between the logarithm of Cche and ΔE in the range including . Therefore, it can be seen that the method of the present invention is effective for measuring cholinesterase.

Example 5 (Analysis of Free Fatty Acid) As an enzyme-immobilized membrane, a film was used in which acyl-CoA synthetase and acyl-CoA oxidase were immobilized and a sparsely soluble magnesium salt was contained. The biochemical analysis sensor shown in FIG. 2 was produced in the same manner as in Example 1 except for the above.

The sensor prepared above was set in the biochemical analysis system described in FIG. 3, and a standard solution with a known free fatty acid concentration was measured in order to create a calibration curve. ATP was added to this standard solution in advance. When making measurements, measure the difference (ΔE) between the measured electrode potential before the enzymatic reaction and the measured electrode potential when the reaction has progressed and reached a steady state value. When ΔE was plotted against the logarithm of free fatty acid concentration (Cffa), the relationship shown in FIG. 10 was obtained.

The results in Figure 10 show that Cf
It is shown that there is a substantially linear relationship between the logarithm of fa and ΔE. Therefore, it can be seen that the method of the present invention is effective for measuring free fatty acids.

[Effects of the Invention] As described in detail above, the present invention provides a compact and simple analysis method that is quick in measurement time and has a low unit cost per measurement item, and has great effects.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view showing one embodiment of the biochemical analysis sensor according to the present invention, FIG. 2 is a cross-sectional view showing another embodiment of the biochemical analysis sensor according to the present invention, and FIGS. 3 and 4 are respectively Second
Figure 5 shows an example of a biochemical analysis system using the sensor shown in Figure 5.
7 to 7 are cross-sectional views showing further embodiments of the biochemical analysis sensor according to the present invention, and FIGS. 8 to 12 show the results of biochemical analysis using the biochemical analysis method of the present invention. It is a graph. 1.11. ...Sensor body 4,15... Measuring electrode, 5.16...Hydrogen supply pipe, 6.2
1... Enzyme immobilization membrane. 17... Cellulose acetate membrane, 18... Basic electrolyte thin layer, 19... Reference electrode, 31.61-69... Biochemical analysis sensor, 32.
51...Flow cell, 34,70.74...Sample tube, 37.73...Reaction tank, 39°79...
・Piping, 40-42.80-83...liquid pump, 4
3.85...Record analysis mechanism, 44°86...Display mechanism, 45.87...Control mechanism. Figure 1 Applicant's agent Patent attorney Takehiko Suzue Figure 2 Figure ΔE (mV) Figure ΔE (mV) Figure ΔE (mV) ffa (uEq/J) Figure

Claims (5)

[Claims] (1) Generate hydrogen peroxide from the substance to be measured through at least one step of enzymatic reaction, quantify the generated hydrogen peroxide, and measure the concentration of the substance to be measured based on the amount of hydrogen peroxide. A biochemical analysis method, in which hydrogen peroxide produced by the enzymatic reaction is brought into contact with a measurement electrode made of a metal having hydrogen storage properties that is sensitive to hydrogen peroxide and outputs a voltage value corresponding to the amount of hydrogen peroxide. A method comprising: quantifying hydrogen peroxide produced in the enzymatic reaction from the output voltage value of the measurement electrode. (2) The method according to claim 1, wherein one or more enzymes necessary for the enzymatic reaction that generates hydrogen peroxide are contained in an enzyme-immobilized membrane provided in close contact with the external surface of the measurement electrode. . (3) A third substance necessary for the enzymatic reaction other than the analyte and the enzyme is added to the enzyme-immobilized membrane and/or
The method according to claim 2, wherein the method is used by being added to a substance to be measured. (4) Generate hydrogen peroxide from the substance to be measured through at least one step of enzymatic reaction, quantify the generated hydrogen peroxide, and measure the concentration of the substance to be measured based on the amount of hydrogen peroxide. A sensor used in a biochemical analysis method, comprising a sensor body made of a hollow cylindrical body, the upper end of which is sealed, and at least a portion of the lower end having an opening, and a hydrogen storage device connected to a lead wire. a measurement electrode made of a metal thin film and provided to seal the lower end opening of the sensor body; a mechanism for maintaining hydrogen pressure inside the sensor body at a predetermined value; A biochemical analysis sensor characterized by having at least one type of enzyme immobilized thereon for generating hydrogen oxide, and comprising an enzyme immobilization membrane provided to cover the external surface of the measurement electrode. . (5) Generating hydrogen peroxide from the substance to be measured through at least one step of enzymatic reaction, quantifying the generated hydrogen peroxide, and measuring the concentration of the substance to be measured based on the amount of hydrogen peroxide. A sensor used in a biochemical analysis method, comprising a sensor body made of a hollow cylindrical body, the upper end of which is sealed, and at least a portion of the lower end having an opening, and a hydrogen storage device connected to a lead wire. a measurement electrode made of a metal thin film and provided to seal the lower end opening of the sensor body; and a measurement electrode provided in communication with the inside of the sensor body in order to maintain the hydrogen pressure inside the sensor body at a predetermined value. an organic polymer thin film permeable to dissolved hydrogen peroxide provided covering the external surface of the measurement electrode; and a hydrogen gas supply pipe provided between the organic polymer thin film and the measurement electrode. a thin layer of basic electrolyte solution; a reference electrode that is in contact with the thin layer of basic electrolyte, maintains insulation from parts other than the basic electrolyte layer, and is connected to an external lead; and a reference electrode that is connected to the substance to be measured. At least one type of enzyme for generating hydrogen peroxide as a starting material is immobilized thereon, and an enzyme immobilization membrane is provided covering the outer surface of the polymer thin film. Biochemical analysis sensor.