DD229222A1 - PROCESS FOR STABILIZING ENZYME ELECTRODES WITH IMMOBILIZED UREASE - Google Patents

Abstract

In the invention, a method for the stabilization of enzyme electrodes with immobilized urease for the quantitative determination of urea is described. The stabilization method consists of adding a complexing agent to the reference electrode electrolyte of a p H combination electrode serving as the electrochemical sensor of the enzyme electrode and storing the immobilized urease electrode in a stabilizing solution prepared by dissolving a nitrogenous buffer substance, a organic acids, a sulfhydryl compound, a complexing agent, a reducing agent and a polyol. Urea electrodes treated in this way are stable in their enzyme activity for a long time under both working and storage conditions.

Description

Field of application of the invention

The invention relates to a method for stabilizing the immobilized urease in potentiometric enzyme electrodes. Areas of application for the invention lie in analytical chemistry, medicine and the agricultural and food industry.

Characteristic of the known technical solutions

The enzyme urease highly specifically splits the substrate urea into the products carbonic acid and ammonia. Because of this property, urease is used in the analytical field for the quantitative determination of urea in physiological solutions by determining the said products of urea reaction by means of optical methods, enzyme test strips or enzyme electrodes. In the case of the enzyme electrodes, the urease is immobilized by suitable methods directly on the surface of an electrochemical sensor. Since the hydrolytic cleavage of urea by the urease is associated with a shift in the pH value, pH glass electrodes are particularly suitable as detection sensors. However, enzyme electrodes in which the urease is immobilized on the pH-sensitive surface of pH glass electrodes, have only a very low working and storage stability because of the inactivation of urease, for example by inhibitors such as the silver ions of the reference electrode of the pH sensor or enzyme aging. As a result, the practical uses are very limited. This is all the more regrettable because the pH measurement technology today belongs to the general technical standard of many analytical laboratories and enzyme electrodes for urea determination on the basis of the pH measuring principle could find a wide application with simple measuring devices.

For soluble urease, various methods have been described for their stabilization, which consisted in the addition of a wide variety of chemicals and biochemicals to the urease preparations (US Patent 4378430, FRD Laid-Open 2612726 and Biochim Biophys Acta 146 (1967) 216). However, these methods are not suitable for stabilizing enzyme electrodes for urea determination, in which the urease is immobilized on the pH-sensitive surface of the electrochemical sensor.

Object of the invention

The aim of the invention is to find a method for the stabilization of enzyme electrodes with immobilized urease in order to improve their potential applications.

Explanation of the essence of the invention

According to the invention the stability of immobilized at pH glass electrodes urease is increased by the electrolyte of the reference electrode with a complexing agent at a concentration of 10 ^-3 -10 ^-5 mol / l is added and the enzyme electrodes are kept in a stabilizing solution which is prepared by dissolving a nitrogen-containing buffer substance in a concentration of 10 ~ ¹ -10 ~ ⁴ mmol / l, an organic acid or its alkali salt in a concentration of 10 ~ ¹ -10 ~ ⁴ mol / l, a sulfhydryl compound in a concentration of 10 ~ ³ -10 " ⁵ mol / l, a complexing agent in a concentration of 10 ~ ³ -10 ~ ⁵ mol / l, a reducing agent in a concentration of 10 ~ ² -10 ~ ⁴ mol / l and a po'yolischen compound in a concentration of 10 ~ ³ ~ 10 ~ ⁵ mol / l in distilled water to give a pH of 6.0-8.0, preferably around the neutral point, of the stabilizing solution that is formed as nitrogen-containing buffer substances tris (hydroxymethyl) aminomethane or triethynolamine are suitable for preparing the stabilizing solution according to the invention, citric acid, tartaric acid or maleic acid as the sulfhydryl compound, mercaptoethanol or thioglycolic acid as sulfhydryl compound, complexing agent for the electrolyte of the reference electrode and for the stabilizing solution ethylenediaminetetraacetic acid or endocyclic heteroaromatic tertiary nitrogen atom such as imidazole or 8-hydroxyquinoline, as a reducing agent sodium sulfite and as a polyolic compound glycerol or dextran. The stabilizing solution is prepared by first dissolving all the substances mentioned except the organic acid in the required concentrations in distilled water and then adjusting the pH to the desired value with the organic acid.

After the reference electrode has been filled with the electrolyte solution containing the complexing agent in dissolved form and the urease has been immobilized on the pH-sensitive surface of the glass electrode, the enzyme electrode is dipped in the stabilizing solution and stored therein until use. Before being used for urea determination, it is washed with the measurement solution and stored again in the stabilization solution after the measurements have been completed. Under these working and storage conditions, the urea electrode, which uses the pH measuring principle directly, is longer than one

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Month stable, while under storage in buffer solutions or aqueous measuring solutions without additives only a stability of 2-3 days is achieved in these electrodes. The storage stability of the urea electrodes is substantially improved by the storage in the stabilizing solution according to the invention. After storage for 3 months, the enzyme electrodes with immobilized urease still have 85-100% of the starting activity. Hereinafter, the subject invention will be explained by examples, but should not limit the scope thereof.

embodiment example 1

A pH electrode is filled with an electrolyte which has been formed from a saturated KCl solution and imidazole in a concentration of 10 ~ ⁴ mol / l, and the pH-sensitive glass surface of the pH electrode, the urease is immobilized by known methods , Then, in one liter of distilled water, dissolve 10 ~ ² moles of tris (hydroxymethyl) aminomethane, 10 ~ ³ moles of mercaptoethanol, 10 " ³ moles of imidazole, 10 ~ ² moles of sodium sulfite, and in this solution add 1 g of dextran until completely dissolved This solution is adjusted to a pH of 7.4 after addition of maleic acid in distilled water (10 ~ ² mol / l) and the enzyme electrode is stored in a part of this stabilizing solution until it is used the electrode still 87% of the initial activity, while a stored in Tris buffer of pH 7.4 and not treated as described type electrode was completely inactive after 5 days.

Example 2

The reference electrode of a pH combination electrode is filled with a saturated KCl solution containing 10 " ⁴ mol / l of ethylenediaminetetraacetic acid, and urease is immobilized on the pH-sensitive surface of the glass electrode according to methods described in the literature triethanolamine (10 ^-2 mol), thioglycolic acid (10 ^-3 mol), ethylenediaminetetraacetic acid (10 ~ ³ mol), sodium sulfite (10 ^-2 mol) and glycerol (10 ^-2 mol) in one liter of distilled water, a solution is prepared which It is adjusted to a pH of 7.0 with a solution of citric acid (10 ~ ² mol) with the urea electrode, 10-20 measurements are taken daily, and in the absence of time it is stored in the described stabilizing solution Month of daily use, an activity of more than 75% compared to the starting activity, while an electrode, the ge without the treatment according to the invention was stored, after 2-3 days no longer possessed activity.

Claims (3)

-1- 695 76 Invention claim: 1. A method for stabilizing enzyme electrodes with urease immobilized on the pH-sensitive surface of a pH electrode, characterized in that as the electrolyte for the reference electrode of the pH electrode, a saturated KCl solution with a therein in a concentration of 10 ~ ³ - 10 ~ ⁵ mol / l dissolved complexing agent is used and the enzyme electrode is stored in the measuring time in a stabilizing solution. 2. The method according to item 1, characterized in that the stabilizing solution is formed by dissolving a nitrogen-containing buffer substance in a concentration of 1O ^-1 -10 ~ ⁴ mol / l, an organic acid or its alkali salt in a concentration of 10 " ¹ -10 ~ ⁴ mol / l, a sulfhydryl compound in a concentration of 10 ~ ³ -10 ~ ⁵ mol / l, a complexing agent in a concentration of 10 ~ ³ -10 ~ ⁵ mol / l, a reducing agent in a concentration of 10 ~ ² - 10 ~ ⁴ mol / l and a polyol compound at a concentration of 10 ~ ³ -10 ~ ^s mol / l in distilled water such that a pH of 6.0-8.0, preferably around the neutral point, is formed Stabilization solution is created. 3. The method according to item 1 and 2, characterized in that as nitrogen-containing buffer substances for the preparation of the stabilizing solution, for example, tris (hydroxymethyl) -aminomethan orTriäthanolamin, as organic acids citric acid, tartaric acid or maleic acid, as sulfhydryl mercaptoethanol or thioglycolic acid, as a complexing agent for the Electrolytes of the reference electrode and for the stabilizing solution of ethylenediaminetetraacetic acid or heteroaromatic compounds with endocyclic, tertiary nitrogen atom such as imidazole or 8-hydroxyquinoline, as a reducing agent sodium sulfite and as a polyolic compound glycerol or dextran.