JPS63149554A - Enzyme sensor - Google Patents

Abstract

PURPOSE:To separate and quantitify a plurality of substrates with a single enzyme sensor, by mounting an enzyme immobilized membrane, an ion exchange membrane and an ion sensor. CONSTITUTION:Enzyme immobilized membranes 23, 24 are polymer membranes immobilizing enzymes respectively specifically reacting with different substrates by a covalent bond and the substrates corresponding to the number of the different enzyme immobilized membranes are detected. An ion exchange membrane 24 has ion exchange capacity to the ion held between the immobilized membranes 23, 25 and well transmits the ion. Further, an ion sensor 27 detects the ion formed by the immobilized membranes 23, 25. Even when the number of substrates increase, the same ions occurring from the different substrates similarly reach the sensor 24 at different times according to the order converted to the ions. This sensor 24 successively detects the same ions converted from the substrates by enzymatic action and can separate and quantify the different substrates.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] This invention relates to the structure of an enzyme sensor that measures multiple substrates.

(Prior art) An enzyme sensor is a biosensor that uses biological functions to detect a specific substrate using an enzyme that has a catalytic function that causes a specific reaction to occur with a specific group '1t (reactive species). It is one of the

Enzyme sensors can specifically detect glucose, urea, creatinine, etc. due to the substrate selectivity of enzymes.

Conventionally, an enzyme sensor has an enzyme-immobilized membrane 23 in which an enzyme such as urease is immobilized on a polymer membrane by a method such as a covalent bonding method or a cross-linking method, and an oxygen electrode 21 and a nitrifying bacteria-immobilized B 22, as shown in FIG. Ammonia ion sensor 27 consisting of
It was constructed by pasting them facing each other. In this conventional example, urea in water is hydrolyzed by the enzyme urease to produce ammonia (Nus) and carbon dioxide (C(h)) as shown in the reaction formula fi+.

Co (NHz)z +H! Otomi 2 N fI 3
+ COz-- (1) The generated NI (3) is converted to ammonia ion (NH, "") in the acidic escort aqueous solution.

The generated NH,'' ions are detected by the ammonia ion sensor 2.
Reacts with oxygen in the water in the nitrifying bacteria immobilization membrane 22 of No. 7,
Nitrite is converted to nitrate. Therefore, oxygen in water is N
Since it decreases in accordance with the amount of H#", the amount of decrease can be detected by the oxygen electrode 21.

In this way, urea in water can be specifically detected as a decrease in the oxygen reduction current of the oxygen electrode 21.

In addition to the conventional enzyme sensor as described above, as shown in FIG. can also be measured at the same time. Creatinine diminase specifically reacts with creatinine and generates NHt'' ions from creatinine.

[Problem that the invention seeks to solve]

However, in the conventional example shown in Figure 3, since the enzyme-immobilized membrane is single, it only reacts with a single substrate, and as a result, multiple types of enzyme sensors are required to detect multiple reactive species. . Furthermore, in the conventional example shown in Fig. 4, since there are multiple enzyme-immobilized membranes, it is possible to respond to multiple substrates;
4" ions diffuse at the same speed, so both Nl(,"
The ions reached the ion sensor at the same time, making it impossible to separate and quantify urea and creatinine.

The purpose of this invention is to make the same ions derived from different substrates reach the ion sensor at different times,
An object of the present invention is to provide a hybrid biosensor that can separate and quantify multiple substrates using a single enzyme sensor.

[Means for solving problems]

In order to achieve the above object, the present invention provides f) a plurality of different enzyme-immobilized membranes 23, 25 that decompose different substrates to generate the same ion; and (b) the immobilized (c) Ion exchange @ 24 which is sandwiched between the membranes 23 and 25 and has an ion exchange ability for the aforementioned ions and allows the aforementioned ions to pass through well; The ion sensor 27 is provided for the purpose of detecting the condition.

[Effect]

The enzyme-immobilized membranes 23 and 25 are polymer membranes in which enzymes that specifically react with different substrates (reactive species) are immobilized by a covalent bonding method, a cross-linking method, etc., and generate the same ions that are detected by the ion sensor. Substrates corresponding to the number of different enzyme-immobilized membranes are detected.

If the ion exchange membrane 24 is a cation exchange membrane, it allows cations to permeate through it more quickly than anions or neutral molecules. In the case of anion exchange membranes, anions pass through them more quickly than cations and neutral molecules. Ions derived from the group 9A generated in the enzyme immobilization membrane 25 enter the ion exchange membrane 24 together with the substrate B that is not converted into ions in the immobilization membrane 25, and permeate there, but the ions are more neutral in the ion exchange membrane. Since the diffusion rate is higher than that of the molecular substrate, ions originating from substrate A pass through the ion exchange 'Mlt, 24 more quickly than substrate B. The ions originating from the substrate A pass through the next enzyme-immobilized membrane 23 as they are and reach the ion sensor 27 . On the other hand, the substrate B passes through the ion exchange membrane 24 due to the ions derived from the group iA and enters the enzyme immobilization membrane 23.
Here, it is converted into the same ion as the ion originating from group 1iA. After passing through the enzyme-immobilized membrane 23, the ions originating from the substrate B reach the ion sensor 24 behind the ions originating from the substrate A.

The above relationship applies in the same way even if the number of substrates increases,
The same ions originating from different substrates reach the ion sensor at different times according to the order in which they are converted into ions.

The ion sensor 24 sequentially detects the same ions converted from a substrate by the action of an enzyme, and separates and quantifies different substrates.

The dialysis membrane prevents the ion sensor and enzyme-immobilized membrane from being contaminated by proteins.

〔Example〕

Next, embodiments of the present invention will be described based on the drawings. 1st
The figure is a schematic cross-sectional view of an enzyme sensor according to an embodiment of the present invention, where 21 is an oxygen electrode, 22 is a nitrifying bacteria immobilized membrane (22A is an immobilized nitrifying bacteria), and the ammonia ion sensor 2
7 is composed. 23 is a urease enzyme immobilized membrane, 24
25 is a Nafion membrane which is a cation exchange membrane, 25 is a creatinine diminase enzyme immobilized membrane, and 26 is a dialysis membrane.

Each membrane is provided in close contact with each other.

Such an enzyme sensor can be manufactured by the following method. The oxygen electrode 21 is provided with a noble metal anode, an internal electrolyte, and a noble metal cathode, and an oxygen permeable membrane such as Teflon is tightly adhered to the surface of the cathode.

The oxygen-permeable membrane retains the internal electrolyte and allows oxygen from the outside to permeate and supply it to the cathode, producing an oxygen reduction current. The cathode is driven at a constant potential.

In the nitrifying bacteria immobilization membrane 22, an aqueous solution containing nitrifying bacteria 22A is dripped onto a porous acetyl cellulose membrane, and the nitrifying bacteria 22A are adsorbed and immobilized onto the membrane. Urease enzyme immobilization membrane 23. The taleatinine diminase enzyme-immobilized membrane 25 has urease and creatinine diminase immobilized on a polymethylglutamate derivative by a covalent bonding method, and has a film thickness of 10 tr.
A film is formed on m.

The ion exchange membrane is 5% of Nafion (cation exchange membrane)
A film is formed to a thickness of 6 fIm using an ethanol solution.

As an ammonia ion sensor, in addition to an ion sensor using microorganisms as in this embodiment, a liquid film type NO sensor using nonaction/mochicution can be used.

Ion sensor, diaphragm-type NH using glass membrane, ion sensor, ion-sensitive field effect transistor (I
SFET) etc. can also be used.

In the enzyme sensor shown in FIG. 1, urea.

Creatinine measurements are carried out in a buffer solution with pH=3.0. Oxygen in the buffer solution reaches the enzyme-immobilized membrane 23.25 together with urea and creatinine, and creatinine is hydrolyzed by creatinine diminase to form ammonia (
NH,) which is converted to N114° ion in an acidic buffer solution. Urea is hydrolyzed by urease to produce Nl+4' ions in the same way. N114
``The ions reach the ammonia ion sensor 27 together with oxygen, and the nitrite ions (
NOt-) + nitrate ion (NO3-). As a result of the above reaction by nitrifying bacteria/I2 element is consumed,
The oxygen reduction current in the oxygen electrode 8i21 decreases. The amount of decrease in oxygen reduction current is proportional to the amount of NH4'' ions.N
H, “The amount of ions is the amount of urea.

Since it is proportional to the amount of creatinine, urea and creatinine can be quantified based on the amount of decrease in oxygen reduction current. Although not shown, the oxygen electrode 21 has an oxygen permeable membrane, through which oxygen in the buffer solution passes and reaches the internal cathode electrode, where it is cathodically reduced and generates an oxygen current.

In the Nafion membrane 24, which is a cation exchange membrane, N
H," ions have a higher diffusion rate than neutral molecules. Creatinine is converted to Nl(," in the taleatininine diminase enzyme-immobilized membrane 25, but urea enters the Nafion membrane 24 as is.N)1. "Ions diffuse more quickly than urea in Nafion G!24, pass through the urease 55 element immobilized 11ff23, and reach the ammonia ion sensor 27. Urea passes through the Nafion membrane later than NH4 ions derived from creatinine. However, it is converted into N114'' ions in the urease enzyme immobilized membrane 23, and reaches the ammonia ion sensor 27 behind the NHa'' ions derived from creatinine. When the oxygen current change Δ■ is plotted against time, the output characteristic diagram shown in FIG. 2 is obtained. The plateau in A is the current reduction due to creatinine, and the plateau in B is the current reduction due to urea and creatinine. The urea signal can be obtained by subtracting the A plateau from the B plateau, and it can be seen that the urea signal lags the creatinine signal by about 5 minutes.

In the above embodiments, creatinine and urea are measured, but amino acids, adenosine, etc. can also be detected separately with an ammonia ion sensor by providing enzyme-immobilized membranes for each.

Furthermore, although an ammonia ion sensor is used in the above embodiment, a carbon dioxide gas sensor may also be used to detect bicarbonate ions in water. In this case, it is also possible to isolate substrates 1 that contain carbon dioxide gas, such as urea, uric acid, amino acids, oxalic acids, etc., using enzymes. As the ion exchange membrane, an anion exchange membrane is used because bicarbonate ion HCO3- is an anion.

〔Effect of the invention〕

According to this invention, (a) a plurality of different enzyme-immobilized membranes that decompose different substrates to generate the same ion; and (c) an ion sensor for detecting the ions generated in the immobilized membrane, so that ions are more easily transmitted than neutral molecules in the ion exchange membrane. Depending on the order in which the substrates are converted into ions for rapid diffusion, ions from each substrate reach the ion sensor in sequence, making it possible to separate and quantify multiple substrates with a single enzyme sensor. becomes.

[Brief explanation of the drawing]

FIG. 1 is a schematic sectional view of an enzyme sensor according to an embodiment of the present invention, FIG. 2 is an output characteristic diagram of an enzyme sensor according to an embodiment of the present invention, and FIGS. 3 and 4 are schematic diagrams of a conventional enzyme sensor. FIG. 23 Niurease enzyme immobilized membrane, 24: Nafion membrane,
25: Creatinine diminase enzyme immobilized membrane, 27:
Ammonia ion sensor. Time (minutes) Figure 2 13rI! J 7 Somoni 71 Onsenbu Suppuration

Claims (1)

[Scope of Claims] 1) (a) a plurality of different enzyme-immobilized membranes that decompose different substrates to generate the same ion; and (b) a membrane that is sandwiched between the immobilized membranes and that reacts with An enzyme sensor comprising: an ion exchange membrane that has ion exchange ability and allows the aforementioned ions to pass through well; and (iii) an ion sensor for detecting the aforementioned ions generated in the immobilized membrane. 2) The enzyme sensor according to claim 1, wherein the plurality of enzyme immobilization membranes are immobilization membranes that generate ammonia ions. 3) The enzyme sensor according to claim 1, wherein the ion exchange membrane is a naphion ion membrane.