Urease immobilizing urea electrode and its production

[Detailed Description of the Invention]

[Application of the Invention] the present invention relating to an enzymatic electrode which measures the urea dissolved in solution, and a manufacturing method for the same, especially quantifying urea for a short time -- good -- it is related with a Bo enzymatic electrode and a manufacturing method for the same. [Description of the Prior Art] The enzyme with high reaction singularity to a substrate (measuring object substance) is fixed, and the enzymatic electrode which combined this, an ion electrode, etc. is developed several years before 1o (G, G.). Quilbault : "Handbook of Enzymatic Ha Method of @nalysis They are 4arcei to ". Dekker. New York The part Different(ed) in the practical use stage in recent years (1976). One of such the enzymatic electrodes has a urea electrode which uses urea as a measuring object substance. The complications which used 7 Lyase for one in it as enzyme, and fixed this although there were some from which some forms differ as a urea electrode. base -- there is a thing with the structure where the film was made to approach a Ammonicum ion selective electrode. it is said that the urea electrode of this form has the long time which measurement of one sample takes -- a problem Ke owner is carried out. [Objects of the Invention] The object of the present invention is to provide a urea electrode which measurement of ■ sample takes and which can carry out all the time short stories, and a manufacturing method for the same in the urea electrode of such a form. [Summary of the Invention] The structure of the conventional urea electrode body is typically shown in Drawing 1. It will become below if the measurement principle of urea by this enzymatic electrode is described briefly. It is decomposed by Urease fixed film 1 and ammonium ion produces the urea contained in sample solution 5. The part of the ammonium ion which arose is measured with the ammonium ion selectivity electrode which comprises ammonium ion induction film 2, internal electrolysis/Vi, 3, and silver salt-ized silver electrode 4. Such an electrode is Analytical, for example. It is indicated to Chem, 49.1067~1078 (1977), etc. In this electrode, the measurement time required is dependent on urea and ammonium ion penetration ability of a Urease fixed film. especially the influence of latter is thick -- it is -- . When artificers' structure of a Urease fixed film is the same, With the chemical nature on the surface of a structure of an ammonium ion penetration capability Urease fixed film, when the thick thing Left and right(ed), especially the structure surface had much quantity of an amino group, ammonium ion penetration ability newly discovered the high thing from the experiment. Therefore, the measurement time required of a urea electrode can be shortened by introducing an amine machine into the structure surface of a Urease fixed film. the main point of the present invention is in using the high Urease fixed film of the penetration ability of ammonium ion, and not only an ammonium ion selectivity electrode but be direct -- be indirect -- of course, that can apply the present invention with the electrode which measures ammonium ion An example of such an electrode is Ana-1ytical. It is indicated to Chem, 49.1067~1078 (1977), etc. [Example] Hereinafter, one example of the present invention is described in detail. Drawing 2 is a lineblock diagram of the body part of the urea electrode used in this example. In a figure, as for 13U enzymatic electrode and 14, a reference electrode and 15 are ground electrodes, As for 1, an ammonium ion induction film, and 6 and 7 are an ion permeation film, and a silver salt-ized silver electrode and the arrow for which as for 4.8 and 9 electrolyte liquid and 16 show a small tube, 17 shows Slow fFJ liquid, and 18 shows the direction of the flow of buffer solution 3.10 and 11 a Urease fixed film and 2. If buffer solution is made small tube 16 with a career and a small quantity (a metaphor is 1omut) passes samples, such as blood, it will be hydrolyzed when Urease fixed film 1 of enzymatic electrode 13 is touched, and ammonium ion will produce the urea contained in a sample. The electrical change for which the membrane potential of ammonium ion induction film 2 was changed, and it depended on the urea concentration in a sample between enzymatic electrode 13 and reference electrode 14 as a result produces ammonium ion. This electrical change is observed as a mountain type change which made the baseline the time of pouring only Slow fiJ liquid in time. It can be regarded as time to carry out few [ of until it returns from the appearance start of Mountains change to base rye /] to measurement of one sample. As Urease solid f-ized film 1, they are Urease (product made from What-ringer) 40 m g, and Albumi/(cow). After dissolving in Seikagaku 80 m g k 0.2 M phosphate buffer solution (pH 7, 4) 1mt and adding a proper quantity of 2.5% glutar r Ruthehyde aqueous g and 280microt to this, After spreading on a glass plate, having settled for more than 10 minutes, carrying out gelling film production and exfoliating from a glass plate, what carried out immersion treatment for 30 second~1 o minutes was used into 0 neutralized with chloride, and 1~10% of ethylene diamine solution. When this Urease fixed film was used, it was also accepted that ' electrode output increases 5~30% compared with the There was case for Urease fixed film Wo which did not carry out immersion treatment in ethylene Diami / solution as an effect which measurement 7vT important point time was shortened 10% or more, and is different. When the example was described concretely and it processed by 2% of ethylene diamine, 1019 films per membrane product 1cnn3 which carry out an amino Motohira owner were obtained. Completion Minot cardinal numbers in a film of an unsettled thing are 1017 piece /Crn3. What measurement of urea nitrogen dark i'loomg/dt took for 70 seconds by an unsettled film was shortened by a processing film at 55 seconds. It replaced with ethylene diamine solution, the above-mentioned Fryase gelling film was immersed in poly lysine solution, and the width side made the effect experimental. As poly lysine, that from which the molecular weight range differs was prepared [ various ], and was used. As a result, the same effect same at processing for a short time as ethylene diamine was accepted by the case where 3000 or less-molecular weight Po IJ IJ resin solution is used. It was a There was case for Vo IJ IJ resin of 1 or less JOOO of molecules that was especially good. When it processed with the 0.5% solution of the poly lysine of average molecular weight 1000, the film which has an amine machine of 10"f[iM/c-n+' was obtained, and it was remarkably shortened compared with 70 God of a A bit unsettled film in time Vj and 50 seconds which measurement of the sample of urea nitrogen concentration 100 mg/d2 takes. Next, various kinds of Ule Completion-Ze 111 Running-ization 11M from which the number of the added amine machine by changing the ethylene diamine processing time of the gelling film of Urey r-Ze differs is prepared, As a result of examining the characteristic as each 1. urea electrode, it is membranous body f (amine more than i 1 cw+3 hit 10'delta Pieces: the film which has I'i- showed the good special order.). in addition -- seven friends' number of measurement described until now immerses a film in fluorescein iso Thiona 2 and - A solution, and performs a fluorescence expense color -- this amount Request of fluorescence -- things performed. albumin solution of each Elm from which the degree of 1 Sentiment differs as a standard sample of the number of amine machines -- heat -- hardening -"It was film was made and what performed the same fluorescence dyeing as this was used. A fixed quantity of the amine machine by this method has about ±20% of error, and although it is difficult to acquire [ which is Ji Jing ] a value absolutely, a digit number can be measured correctly. Although the above-mentioned example described examples, such as ethylene Cyamine and lysine, the substance which otherwise has two or more amino groups in one molecule, such as Quanidine, -- the structure surface of a Urease fixed film -- what crosses generally and adds an amine machine showed the effect same for adopting the same way of using as ethylene diamine as Yo Si. [Effect of the Invention] The measurement time required of - sample can be shortened in the present invention. Therefore, at least single , has in time the effect that many samples can be measured. the present invention -- getting twisted -- since an electrode output increases, there is an effect of the improvement in sensitivity.

[Brief Description of the Drawings]

The mimetic diagram in which Drawing 1 shows the structure of a urea electrode body, and Drawing 2 are key maps showing the composition of a urea electrode. 1 ... Urease fixed film, 2 ... an ammonium ion induction film and 3, 10.11 ... electrolyte liquid, 4, and 8*9 [ ... A reference electrode, 15 / ... A ground electrode, 16 / ... Small tube ] ... A silver salt-ized silver battery, 6, 7 ... An ion permeation film, 13 ... An enzymatic electrode, 14 Y 1 figure Certain 2 figure