WO2012133633A1

WO2012133633A1 - Disposable lysine sensor

Info

Publication number: WO2012133633A1
Application number: PCT/JP2012/058334
Authority: WO
Inventors: 武司上村; 希稲葉; 加成恵板岡; 悦夫篠原
Original assignee: 株式会社テクノメデイカ
Priority date: 2011-03-29
Filing date: 2012-03-29
Publication date: 2012-10-04

Abstract

[Problem] To provide a disposable sensor capable of measuring lysine. [Solution] The disposable lysine sensor relating to the present invention is a sensor for measuring the matrix concentration of a sample solution by forming an electrode system having at least a measuring electrode and a counter electrode on an electrically insulating substrate and using the electrode system to electrochemically detect changes in the substance concentration during reaction of an enzyme, electron receptor and sample solution, which is characterized by the formation, on the measuring electrode, of a reaction layer that supports lysine oxidase and ferricyan ions, which react with lysine in the presence of the lysine oxidase as a catalyst.

Description

Disposable lysine sensor

The present invention relates to a disposable sensor for measuring lysine.

In recent years, an increasing number of patients become so-called modern malnutrition, which is malnourished despite taking a meal.
Modern malnutrition refers to malnutrition caused by a lack of ingredients necessary for the body due to a bias in the content of the meal, even though a certain amount of meal is ingested.
In the present age when you can choose and eat whatever you like, there are many patients who suffer from modern malnutrition without subjective symptoms due to gastronomy and unbalanced diets.
There are many components necessary for the body, but proteins that play a role in creating muscles, organs, bones, skin, blood, hormones, enzymes, immune substances, etc. are particularly important.
Proteins are macromolecules made by combining 20 amino acids, and the human body is composed of about 100,000 types of proteins. Of these amino acids, 9 types cannot be synthesized in the body, so they must be taken from food, and these 9 types of amino acids are called essential amino acids.

JP-A-10-197473

Lysine, which is one of the essential amino acids, is an important amino acid that repairs body tissues and participates in the growth of the body. If it is deficient, it may cause growth disorders.
As described above, lysine is the most important amino acid even though it is a very important amino acid.
Lysine is not so much contained in vegetable protein, but is abundant in dairy products such as milk and cheese, and soy, meat, and fish. If it is not ingested, it will be deficient in the body, resulting in modern malnutrition.
As mentioned above, lysine is an important item in diagnosing modern malnutrition, but malnutrition is usually diagnosed by examining the amount of albumin. There is no lysine measurement for the diagnosis.
Conventionally, instead of using an enzyme as an electron acceptor, a biosensor using a metal complex such as potassium ferricyanide or an organic compound as an electron acceptor has been proposed. In this type of biosensor, the substrate to be measured and the enzyme The substrate concentration is obtained from the oxidation current value by oxidizing the reduced form of the electron acceptor generated by the enzymatic reaction with the electrode at the electrode (see Patent Document 1). In this type of biosensor, There is no biosensor for measuring lysine.
The inventors focused on the above-mentioned present situation, and conducted intensive research on a biosensor capable of measuring lysine, and found an optimal combination of lysine oxidase and electron acceptor for measuring lysine in a disposable sensor. .
An object of the present invention is to provide a disposable sensor capable of measuring lysine.

In order to achieve the above object, a disposable lysine sensor according to the present invention forms an electrode system having at least a measurement electrode and a counter electrode on an electrically insulating substrate, and reacts with an enzyme, an electron acceptor and a sample solution. In a sensor that electrochemically detects a change in substance concentration with the electrode system and measures the substrate concentration of the sample solution, lysine oxidase and ferricyan ion that reacts with lysine using the lysine oxidase as a catalyst are held on the measurement electrode. The reaction layer is formed.
The lysine oxidase can be composed of, for example, a mold-derived enzyme, specifically, an enzyme derived from a Trichoderma bacterium, and more specifically, an enzyme derived from Trichoderma viridae.
Alternatively, a second measurement electrode may be provided on the electrically insulating substrate, and a reaction layer not containing lysine oxidase holding ferricyanide that reacts with lysine using lysine oxidase as a catalyst may be formed on the second measurement electrode. Good.
Furthermore, a second measurement electrode is provided on the electrically insulating substrate, and a reaction layer holding a protein having no lysine oxidase activity and a ferricyan ion that reacts with lysine using lysine oxidase as a catalyst is formed on the second measurement electrode. May be. In this case, the protein having no lysine oxidase activity may be, for example, inactivated lysine oxidase.

The disposable lysine sensor according to the present invention forms an electrode system having at least a measurement electrode and a counter electrode on an electrically insulating substrate, and electrochemically changes a substance concentration during reaction of an enzyme, an electron acceptor and a sample solution. In the sensor that detects the substrate concentration of the sample solution by detecting with the electrode system, a reaction layer holding lysine oxidase and ferricyanide that reacts with lysine using the lysine oxidase as a catalyst is formed on the measurement electrode. Therefore, lysine can be easily measured. This makes it possible to measure lysine even in diagnosis in general internal medicine and the like, so that more accurate diagnosis of malnutrition can be performed.
The inventors use an enzyme derived from mold as the lysine oxidase, specifically an enzyme derived from Trichoderma genus, more specifically an enzyme derived from Trichoderma viridae, and electron accepting. It was confirmed that favorable results were obtained by using ferricyan ion as a body.
Further, by providing a second measurement electrode on the electrically insulating substrate, and forming a reaction layer not containing lysine oxidase holding ferricyan ion that reacts with lysine using lysine oxidase as a catalyst on the second measurement electrode. Since the amount of lysine can be measured based on the measurement results of the first measurement electrode containing lysine oxidase and the second measurement electrode not containing lysine oxidase, more accurate measurement is possible.
In addition, a second measurement electrode is provided on the electrically insulating substrate, and a protein having no lysine oxidase activity (for example, inactivated lysine oxidase) and ferricia that reacts with lysine using lysine oxidase as a catalyst. By forming the reaction layer holding the ions, the conditions of the first measurement electrode and the second measurement electrode can be made closer, so that more accurate measurement is possible.

It is a figure which shows the measurement principle in the disposable lysine sensor which concerns on this invention. It is a graph which shows the cyclic voltammogram obtained by the cyclic voltammetry method using the lysine oxidase derived from ferricyan ion and mold. (A) is a graph which shows the change of the electric current value when adding lysine sequentially, (b) is a graph which shows the electric current value according to the density | concentration change by addition of lysine. In FIG.3 (b), it is the graph which showed the lysine low concentration side. 1 is a schematic development view of a disposable lysine sensor according to the present invention. (A)-(e) is the schematic which shows the manufacturing method of a disposable lysine sensor. (A) is a schematic top view of a disposable lysine sensor and a portable analyzer, (b) is a schematic top view which shows the state which mounted | wore the portable analyzer with the disposable lysine sensor. It is a block diagram which shows roughly the internal process of a portable analyzer.

Hereinafter, embodiments of the disposable lysine sensor according to the present invention will be described with reference to the accompanying drawings.

FIG. 1 is a diagram showing a measurement principle in a disposable lysine sensor according to the present invention.
As shown in the drawings, the disposable lysine sensor according to the present invention uses lysine oxidase and ferricyan ion as an electron acceptor. Then, lysine as a substrate contained in blood is oxidized by lysine oxidase, and by transferring electrons to ferricyan ion as an electron acceptor via lysine oxidase, ferricyan ion is reduced and ferrocyan ion And the concentration of lysine is measured based on the oxidation response current that flows when ferrocyanic ions are oxidized on the working electrode.

Measurement of lysine (Figure 2)
Used equipment:
・ Potentiot Galvano Stat (HA-501) (made by Hokuto Denko Corporation)
-Function generator (HB-104) (made by Hokuto Denko Corporation)
Measurement condition:
・ Platinum wire is used for both counter and working electrodes ・ Lysine concentration is 5μmol / dL
・ Felicyan ion concentration is 5mM
-Potential scanning range -300 to 800mV
・ Scanning speed 50 mV / s
・ Ambient temperature 28 ℃
In FIG. 2, curve a shows the potential (V) and current density (A / cm 2) when 5 mM ferricyanide was deposited on the working electrode and a lysine solution having a concentration of 5 μmol / dL was added thereto. Showing the relationship.
In FIG. 2, curve b shows the potential when 5 mM ferricyanide is deposited on the working electrode, and a 5 μmol / dL lysine solution and 1 U / ml fungal lysine oxidase are added thereto. The relationship between (V) and current density (A / cm 2) is shown. Here, as a lysine oxidase, a lysine oxidase derived from Trichoderma viridae (EC number: 1.4.3.14 sold by Sigma-Aldrich Japan Co., Ltd.), which is an enzyme derived from mold, was used.
From the curves a and b, when the reaction proceeds, ferricyan ions are consumed in the vicinity of the electrode to generate ferrocyanide ions. By adding lysine oxidase to this, ferricyan ions function as a mediator of lysine oxidase, It can be confirmed that the current value on the oxidation side is increased because ferrocyanic ions are generated. Thus, the redox peak derived from ferricyan ion could be confirmed in the lysine system as well as the glucose system. From this result, it was confirmed that the ferricyan ion functions as an electron acceptor of lysine oxidase.
Curves c and d show cyclic voltammograms by a reaction that does not depend on ferricyan ion (by hydrogen peroxide), respectively. From the curves c and d, it can be confirmed that hydrogen peroxide generated in this reaction does not greatly affect the current value on the oxidation side.

Next, FIG. 3 (a) and FIG. 3 (b) show the results of experiments conducted to confirm what kind of current response is observed in the reaction system of lysine and lysine oxidase as the lysine concentration is increased.
The measurement conditions are as follows.
Used equipment:
・ Potentiot Galvano Stat (HA-501) (made by Hokuto Denko Corporation)
-Function generator (HB-104) (made by Hokuto Denko Corporation)
Measurement condition:
・ Use a gold plate on the counter electrode and a gold-deposited plate on the working electrode ・ Add 22.4 μl of lysine oxidase ・ Ferician ion concentration is 5 mM
-Potential scanning range 550mV
・ Ambient temperature 28 ℃
On the working electrode, ferricyanide at a concentration of 5 mM was deposited, 22.4 μl of lysine oxidase was added thereto, and a lysine solution was added as shown in Table 1. Here, as a lysine oxidase, a lysine oxidase derived from Trichoderma viridae (EC number: 1.4.3.14 sold by Sigma-Aldrich Japan Co., Ltd.), which is an enzyme derived from mold, was used.

FIG. 3A shows a change in current value when lysine is sequentially added, and FIG. 3B shows a current value according to a change in concentration due to the addition of lysine.
As shown in FIGS. 3A and 3B, it was confirmed that the current value increased stepwise as the amount of lysine added was increased.
FIG. 4 is an enlarged graph of 150 nmol / ml or less assumed to be a practical concentration region in the test results shown in FIG.
As shown in the test results of FIGS. 3 and 4, the current value increased stepwise as the amount of lysine added was increased, so that a current response due to the reaction between lysine and lysine oxidase could be confirmed. Moreover, it has confirmed that the electric current value was correlated with the addition amount of lysine.

Next, an example of a disposable lysine sensor to which the above-described measurement principle is applied will be described.
FIG. 5 is a schematic development view of a disposable lysine sensor according to the present invention, and FIGS. 6A to 6E are schematic views showing a manufacturing method of a disposable lysine sensor.
Reference numeral 1 in the drawing denotes an insulating substrate. On this board | substrate 1, after printing gold | metal with a semiconductor printing technique, the 1st measurement pole 2 and the 2nd measurement pole 3 were formed by drying by heating, and silver / silver chloride was printed with the semiconductor printing technique. Thereafter, the counter electrode 4 is formed by heating and drying. The first measurement electrode 2 and the second measurement electrode 3 are formed at positions that are symmetrical with respect to the counter electrode 4. (FIG. 6A).
Next, a photoresist film 8 is formed so as to cover portions of the substrate 1 other than the

electrode portions

2a, 3a, 4a and the

terminal portions

2b, 3b, 4b of the first measurement electrode 2, the second measurement electrode 3, and the counter electrode 4. Provided (FIG. 6B).
Thereafter, a polymer solution in which lysine oxidase, a surfactant, and ferricyan ions are dissolved is applied and dried on the electrode 2a of the first measurement electrode 2, and the first reaction layer 6 is formed on the electrode 2a of the first measurement electrode 2. Form. At the same time, on the electrode 3a of the second measuring electrode 3, the same amount of the polymer solution in which the same amount of the surfactant and ferricyan ion as the first reaction layer 6 is applied is dried. A second reaction layer 7 is formed on 3a (FIG. 6C).
Next, a spacer 9 provided with a notch 9a for forming a specimen channel 12 is disposed on the photoresist film 8 (FIG. 6D), and a cover having an air hole 10 formed thereon. The disposable lysine sensor is completed by providing 11 (FIG. 6E).
A notch 9 a of the spacer 9 surrounds the

electrode portions

2 a, 3 a, 4 a of the first measurement electrode 2, the second measurement electrode 3 and the counter electrode 4, and the air hole 10 of the cover 11, and is arranged on the substrate 1. In this state, the end portion located on the opposite side of the

terminal portions

2b, 3b, and 4b is opened to form the sample introduction port 13. The spacer 9 has a thickness that forms a specimen flow path 12 having a height that causes the capillary action of the specimen between the photoresist film 8 and the cover 11, specifically, for example, about 0.3 mm. It is thickness.
With the configuration described above, when the user introduces a sample such as blood from the sample introduction port 13, the sample is subjected to capillary action in the sample channel 12, and the

electrode portions

2 a and 3 a of the first measurement electrode 2 and the second measurement electrode 3. Will go on.

The disposable lysine sensor configured as described above is used by being mounted on a portable analyzer, for example.
7A is a schematic top view of a disposable lysine sensor and a portable analyzer, FIG. 7B is a schematic top view showing a state in which the disposable lysine sensor is mounted on the portable analyzer, and FIG. 8 is a portable analyzer. It is a block diagram which shows roughly the internal process of an apparatus.
In the figure, symbol A indicates a disposable lysine sensor, symbol B indicates a portable analyzer, symbol 21 indicates a housing of the portable analyzer, symbol 22 indicates a display provided on the upper surface of the housing 21, Reference numeral 23 denotes an operation switch provided on the upper surface of the housing 21, reference numeral 24 denotes a sensor insertion opening provided at one end of the housing 21, and reference numeral 25 denotes an output terminal.
FIG. 8 is a block diagram schematically showing the internal processing of the portable analyzer.

Reference numerals

26, 27, and 28 denote terminals connected to the

terminal portions

2b, 3b, and 4b of the disposable lysine sensor, and reference numeral 29 denotes A control device for calculating the amount of lysine based on input signals from the

terminals

26, 27 and 28, reference numeral 30 denotes a storage device, reference numeral 22 denotes the display, and reference numeral 25 denotes the output terminal.

Hereinafter, a method of using the disposable lysine sensor configured as described above will be briefly described.
As shown in FIG. 7B, the user inserts the prepared disposable lysine sensor A into the insertion port 24 of the portable analyzer B. Thus, the

terminal portions

2b, 3b, 4b of the disposable lysine sensor A are connected to the

terminals

26, 27, 28 provided inside the portable analyzer B.
In this state, when the user stabs his / her finger with a needle or the like to cause bleeding, and the blood is brought into contact with the sample introduction port 13, the blood is sucked into the sample flow path 12 by capillary action, and the first measurement electrode 2. And move to the

electrode portions

2 a and 3 a of the second measuring electrode 3, that is, the reaction layers 6 and 7.
At the first measurement electrode 2, lysine in the sample solution and contaminants in the sample solution are detected.
Further, since the reaction layer 7 does not contain lysine oxidase, only the contaminants in the sample solution are detected at the second measurement electrode 3.
The detection results detected by the first measurement electrode 2, the second measurement electrode 3 and the counter electrode 4 of the disposable lysine sensor A are transmitted through the

terminal portions

2b, 3b and 4b to the

terminals

26, 27, The amount of lysine is calculated by calculating the difference between the detection result in the reaction layer 6 (lysine and contaminants) and the detection result in the reaction layer 7 (contamination matter). And the calculation result is stored in the storage unit 30 and the calculation result is displayed on the display 22 as necessary. The control device 29 is configured to be able to output data stored in the storage unit 30 from the output terminal 25 to an external device as necessary.
The control device 29 can also be configured to determine whether or not malnutrition is based on the measured amount of lysine and output the determination result.

As described above, it has been confirmed that ferricyan ion functions as an electron acceptor of lysine oxidase, and that the current value is correlated with the amount of lysine added in the reaction system of lysine and lysine oxidase. Therefore, as in the above-described example, a lysine oxidase, a surfactant, and ferricyan ion are provided on the first measurement electrode, and the lysine concentration is determined with a simple disposable sensor configured to guide the specimen to the first measurement electrode. There is an effect that it becomes possible to measure.

In the embodiment described above, since the second reaction layer 7 containing the same amount of surfactant and ferricyan ion as the first reaction layer 6 is formed on the electrode 3a of the second measurement electrode 3, the measurement is performed. At this time, the conditions of the first measurement electrode 2 and the second measurement electrode 3 are aligned, so that an accurate measurement result can be obtained.

In the embodiment described above, the second reaction layer 7 containing the same amount of surfactant and electron acceptor as the first reaction layer 6 is provided on the electrode 3a of the second measurement electrode 3, but the second reaction The structure of the layer 7 is not limited to this example. For example, in order to make the conditions with the first reaction layer 6 more uniform, the same amount of surfactant, electron acceptor, and deactivation as the first reaction layer 6 are used. You may form by apply | coating and drying the polymer solution which dissolved the made lysine oxidase.

Furthermore, in the above-described embodiment, the second measurement electrode 3 is provided on the substrate and the second reaction layer 7 is formed on the second measurement electrode 3, but the second measurement electrode 3 is not an essential component. It is not necessary to provide it. For example, by holding a reagent capable of erasing the contaminant contained in the sample on the first measurement electrode 2, the contaminant contained in the sample can be removed, so the second measurement electrode 3 It is possible to obtain a measurement result that is not affected by contaminants even without providing a filter. Examples of contaminants that have a large influence on the measurement of lysine include L-ascorbic acid. In this case, ascorbate oxidase is provided on the first measurement electrode 2 as a reagent for removing L-ascorbic acid. obtain.

In the above-described examples, lysine oxidase derived from Trichoderma viride (EC number: 1.4.3.14 sold by Sigma-Aldrich Japan Co., Ltd.) is used as the lysine oxidase. Without being limited to the examples, any enzyme can be used as long as the ferricyan ion functions as an electron acceptor.
Specifically, mold-derived lysine oxidase other than Trichoderma viridae may be used, and lysine oxidase other than mold-derived, specifically, for example, extracted from immune tissue coming out of fish skin It may be lysine oxidase or human lysine oxidase.

A Disposable lysine sensor 1 Substrate 2 First measurement electrode 2a Electrode 2b Terminal part 3 Second measurement electrode 3a Electrode 3b Terminal part 4 Counter electrode 4a Electrode 4b Terminal part 6 First reaction layer 7 Second reaction layer 8 Photoresist film 9 Spacer 9a Notch 10 Air hole 11 Cover 12 Sample flow path 13 Sample inlet

B Portable analyzer 21 Housing 22 Display 23 Operation switch 24 Sensor insertion port 25 Output terminal 26 Terminal 27 Terminal 28 Terminal 29 Control device 30 Storage device

Claims

Forming an electrode system having at least a measurement electrode and a counter electrode on an electrically insulating substrate;
In a sensor for electrochemically detecting a change in substance concentration during reaction of an enzyme, an electron acceptor and a sample solution with the electrode system and measuring a substrate concentration of the sample solution,
A disposable lysine sensor characterized in that a reaction layer holding lysine oxidase and ferricyan ion that reacts with lysine using the lysine oxidase as a catalyst is formed on the measurement electrode.
The disposable lysine sensor according to claim 1, wherein the lysine oxidase comprises an enzyme derived from mold.
The disposable lysine sensor according to claim 2, wherein the lysine oxidase is an enzyme derived from Trichoderma spp.
The disposable lysine sensor according to claim 3, wherein the lysine oxidase is an enzyme derived from Trichoderma viridae.
A second measurement electrode is provided on the electrically insulating substrate, and a reaction layer not containing lysine oxidase holding ferricyanide that reacts with lysine using lysine oxidase as a catalyst is formed on the second measurement electrode. The disposable lysine sensor according to any one of claims 1 to 4.
A second measurement electrode is provided on the electrically insulating substrate, and a reaction layer holding a protein having no lysine oxidase activity and ferricyan ion that reacts with lysine using lysine oxidase as a catalyst is formed on the second measurement electrode. The disposable lysine sensor according to any one of claims 1 to 4, characterized in that:
The disposable lysine sensor according to claim 6, wherein the protein having no lysine oxidase activity is inactivated lysine oxidase.
The disposable lysine sensor according to any one of claims 1 to 4, wherein a reagent for removing contaminants in the sample liquid is held in the reaction layer formed on the measurement electrode.
The disposable lysine sensor according to claim 8, wherein the contaminant is L-ascorbic acid, and the reagent for removing the contaminant is ascorbate oxidase.