JPS62222158A - enzyme sensor - Google Patents

Abstract

PURPOSE:To effect easy detection of sugarand protein and quantitative measurement, even if anionic disturbing elements, such as phosphate, are existing in common, by installing an anionic film which covers a pH detecting unit and an enzyme-fixing film. CONSTITUTION:An enzyme-fixing film 15 is installed in front of a detecting unit 13 in a pH detecting member 11 and further, an anionic film 17 is installed in such a way that it covers the detecting member 11 and fixing film 15 in the front. If the member 11 is dipped into urine, phosphatic ions are screened by a film 17 preventing it from inside instruction. On the other hand, sugar being of natural characteristic ionically, it is not screened by the film 17 and it reaches the film 15. The sugar is decomposed to gluconic acid by glucose oxidase of the film 15 and the pH value changes. At this moment, the phosphate representing a disturbing element is screened bit the film 17, and consequently, the pH change is detected accurately by the detecting unit 13. Consequently, even under co-existence with the disturbing elements, such as the phosphate, measurement of concentricities of sugar, protein, etc., can easily be performed.

Description

[Detailed Description of the Invention] Technical Field The present invention relates to an enzyme sensor that can easily measure sugar, protein, etc. in urine or a bioreactor by utilizing the selective catalytic action of an enzyme, and particularly relates to an enzyme sensor that can easily measure sugar, protein, etc. in urine or a bioreactor. Regarding the structure of the detection unit.

Traditionally, the test strip method has often been used to detect sugar and protein in urine. However, this method has problems such as being qualitative and requiring skill in determining the results of one judgment.

Additionally, devices that mechanize this determination part are commercially available, but they are expensive.

On the other hand, as a method for analyzing sugar and protein in urine, a method is known that uses immobilized enzymes in combination with an oxygen electrode or the like. Taking sugar as an example, it uses a reaction in which glucose is oxidized by glucose oxidase to produce gluconic acid and hydrogen peroxide.

Generally, the amount of oxygen consumed for sugar decomposition is determined using an oxygen electrode, or the amount of hydrogen peroxide produced is determined using a hydrogen peroxide electrode.

However, this method requires precise analytical equipment because it is necessary to measure trace amounts of oxygen or hydrogen peroxide, and these detection units are easily affected by temperature, so it is difficult to maintain a constant measurement environment. Processing was necessary and troublesome.

It has been reported that a method for detecting sugar in pure water involves combining a pl+ electrode and a glycolytic enzyme, measuring gluconic acid produced by the decomposition of sugar with the pl+ electrode, and quantifying sugar ("Meter"). Sou JlVol 27. (7).

P34-39 (1,984), Ao Maeda/Yoshio Hanasato “
``Composite detection type biosensor element and its application concept''). According to this method, since measurement can be performed using a pH electrode, it is easy to handle, and the measuring device itself is simplified and inexpensive.

However, when attempting to detect sugar in urine using this method, detection was impossible due to interference by phosphates contained in the urine. That is, due to the pH buffering effect of phosphate, even if gluconic acid was produced by decomposition of glucose, it could not be accurately detected as a change in ρ11. This situation also applies to bioreactors. In bioreactors, a buffering agent such as phosphate is usually added in order not to deviate from the optimum ρ1 value as the reaction progresses.

If phosphate is removed by precipitation or column adsorption prior to measuring ρI+, it is possible to measure changes in pH due to the production of gluconic acid, but these pretreatments are extremely complicated. Yes, it is not practical.

Purpose of Clarity - The present invention provides an enzyme sensor that can easily detect and quantify sugars, proteins, etc. using an easy-to-handle pH electrode, even when anionic interfering components such as phosphates coexist. It provides:

[Two blades] (7 [Consideration) The enzyme sensor of the present invention has a pl+ detection section and an enzyme-immobilized membrane provided in close proximity to the pl+ detection section, and the enzyme-immobilized membrane detects a specific component in the liquid to be detected. to convert it into other substances,
An enzyme sensor configured to detect a change in pH due to this conversion using the pH detection section, wherein an anionic membrane is provided to enclose the pH detection section and the enzyme immobilization membrane. Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

FIG. 1 is a cross-sectional view showing an example of the configuration near the detection section of the enzyme sensor of the present invention. An enzyme immobilization membrane 15 is provided on the front surface of the detection section 13 of the pH detection member 11, and an anionic membrane 17 is further provided on the front surface so as to cover the detection section 13 and the enzyme immobilization membrane 15. , these are fixed to the pH detection member 11 by a presser ring 23 via a circle 21. Here, "provided so as to be encapsulated" means that when the PH detection part is immersed in the liquid to be detected, the enzyme-immobilized membrane and the detection part are not directly exposed to the liquid.

This means that an anionic film is provided on the front surface of these to protect them, and it does not matter whether they are actually wrapped.

As the Pi+ detection member 11, a p++ glass electrode is known and commonly used as a general-purpose product, but a pH-ISFET [Ion 5ensit] using a semiconductor element is used.
j, veFeild Effect Transist
(abbreviation for "or") (ion-sensitive field effect transistor)], any device that can detect a change in pl+ can be used.

The enzyme-immobilized membrane 15 can be produced by, for example, a covalent bonding method, a crosslinking method, an entrapping method, or the like. The enzyme is appropriately selected depending on the measurement target, and the conversion results in ρ11
In order to cause a change, a substance having an activity of converting the Kl'l constant target into another substance by decomposition or the like is used. For example, when sugar is the i11'l target, a glycolytic enzyme such as glucose oxidase is used, which converts glucose to gluconic acid, causing a pH change.

Furthermore, when the measurement target is a protein, by using protease, pepsin, or the like as an enzyme, the protein is changed into an amino acid, and po is changed. Membranes based on glass fiber to which glucose oxidase is covalently bonded are commercially available and can also be used for sugar detection.

Anionic membranes become anionic in water and inhibit the passage of ions with similar charges, such as phosphate ions. On the other hand, sugars and proteins dissolved in water have almost no ionicity, so they are not hindered by the anionic membrane.
You can pass this. Ionically neutral substances such as sugars and proteins are thought to diffuse and permeate into anionic membranes through water.

Such an anionic membrane can be obtained, for example, by using polyvinyl alcohol as the membrane material and adding an anionic surfactant thereto. Here, the anionic surfactant is 5-linear alkylbenzene sulfonate (L
AS), α-olefin sulfonate (AO3), alkyl sulfur M salt (AS), soap, etc., and especially CA
S shows a good shielding effect against phosphates. Moreover, sodium salts are typical of these salts. (a
) The ratio of membrane material and (b) anionic surfactant is (a
)/(b)=8/2-515 (dry weight ratio) is preferred. As the amount of anionic surfactant increases, the effect of shielding phosphate anions becomes better, but if too much is added, the strength of the film will be lost, which is not preferable.

Next, a specific example of manufacturing an anionic film will be described.

10% polyvinyl alcohol with a degree of esterification of 90% or more
The mixture diluted with water to about νt% and an anionic surfactant are mixed, then dried until the water content is about 25 to 35%, and then crosslinked with a crosslinking agent such as glutaraldehyde. Crosslinking was carried out under saturated sodium chloride to suppress dissolution of polyvinyl alcohol, and Pl+ was added to initiate the reaction.
The temperature is adjusted to 2 to 3 and carried out at a temperature of 40°C or higher for 5 to 30 minutes.

At this time, the higher the temperature, the faster the crosslinking reaction. If the crosslinking reaction progresses too much, the anionic film that is produced becomes hardened, brittle, and easily torn.

When the detection member 11 shown in FIG. 1 is immersed in urine, phosphate ions are blocked by the anionic membrane 17 and do not penetrate into the interior thereof. On the other hand, since sugar is ionically neutral, it is not shielded by the anionic membrane 17.
It passes through this and reaches the enzyme-immobilized membrane 15. At this time, the anionic membrane contains water and is in a swollen state, and it is thought that the sugar diffuses into the enzyme-immobilized membrane 15 through this water.

N (glucose) is decomposed into gluconic acid by glucose oxidase in the enzyme-immobilized membrane 15, and as a result, p
H value changes. At this time, since the interfering component phosphate is blocked by the anionic film 17 and does not exist, the pH detection section 13 can reliably and accurately detect the change in pl+.

The amount of sugar in the urine can be determined from the pH change caused by gluconic acid produced by the enzymatic reaction and from the analytical m-line prepared in advance by changing the N concentration. human urine is P
Since the range of H5 to H8 is considered to be a normal value, by preparing several types of calibration curves in this range, the Na content in urine can be determined from the change value of Pll. In practical terms.

It is desirable to simultaneously immerse a reference pH detection section for detecting the initial ρ11 and a pH detection section for measuring Pl+ change in urine, and convert it into a sugar concentration using the above method.

In the above explanation, we have explained the method for measuring sugar concentration in urine, but the method is similar for measuring other components such as protein in urine, and depending on the enzyme reaction to be used, an appropriate enzyme-immobilized membrane is used. It would be better if you adopt it.

The same applies to detection samples other than urine. For example, in bioreactors, buffers such as phosphate are added to keep Pll at a predetermined value, but according to the present invention, it is possible to measure Na content etc. without being affected by these interfering components. I can do it.

Effects of the Invention According to the present invention, the concentration of sugars, proteins, etc. can be easily measured using a relatively easy-to-use and inexpensive ρ11 detection device even in the presence of interfering components such as phosphates. I can do it. Therefore, it is possible to easily measure data necessary for health management, such as sugar and protein in urine, and is particularly useful for health management at home. Furthermore, it can be easily detected even at low concentrations that cannot be quantified using conventional test strips, and is useful for early detection of diseases.

Furthermore, the sugar concentration in the bioreactor can be easily determined using al.
It is also industrially useful because it can be determined.

Example 1 containing 70 parts by weight of PVA (degree of esterification 90% or more)
A 50% aqueous solution containing 30 parts by weight of LAS-Na was added to the 0% aqueous solution, and then dried until the water content was about 30% by weight. Next, add 5 glutaraldehyde
Add part by weight, heat at 50°C for 10 minutes to crosslink,
An anionic membrane was obtained.

An enzyme-immobilized membrane on which glucose oxidase was immobilized was placed on a pH glass electrode, and an anionic membrane was further placed on top of that and fixed with an O-ring to create an enzyme sensor as shown in FIG. 1.

When this enzyme sensor (measuring electrode) and a reference pH glass electrode were immersed in sample urine, the initial pH value was 6.0, while the enzyme sensor measured a pH change of 0.6. Ta. From these values, the sugar concentration was determined using a calibration curve prepared in advance and found to be 0.5 gIQ.

On the other hand, a comparative enzyme sensor was prepared in the same manner as above except that the anionic membrane was not used, and when it was immersed in urine, no change in pH could be detected. After removing phosphate through a column filled with ion exchange resin (anion exchange resin: Murofook 1-X8, manufactured by Muromachi Kagaku Kogyo@), the enzyme sensor of a comparative example without an anion film formation was placed in the sample. When the urine sample was immersed in water and measured, a pH change of 0.13 was measured, which was in good agreement with the above value.Furthermore, when the sugar concentration in the urine sample was measured using a combination of glucose oxidase and an oxygen electrode, it was found that 0.5 g. /
Q, which was in good agreement with the value measured by the enzyme sensor of the present invention.

[Brief explanation of drawings]

FIG. 1 is a sectional view showing the vicinity of the detection section of an example of the structure of the enzyme sensor of the present invention.

Claims (1)

[Claims] 1. It has a pH detection part and an enzyme-immobilized membrane provided adjacent to the pH detection part, and the enzyme-immobilized membrane converts a specific component in the liquid to be detected into another substance, and the reaction associated with this conversion An enzyme sensor configured to detect a change in pH using the pH detection section, characterized in that an anionic membrane is provided to enclose the pH detection section and the enzyme immobilization membrane. Enzyme sensor.